Semi-soft c-class immunostimulatory oligonucleotides

ABSTRACT

The invention relates to specific C-Class semi-soft CpG immunostimulatory oligonucleotides that are useful for stimulating an immune response. In particular the oligonucleotides are useful for treating allergy, such as allergic rhinitis and asthma, cancer and infectious disease, such as hepatitis B and hepatitis C.

RELATED APPLICATION

This application is a divisional of U.S. Application Ser. No.11/255,100, filed Oct. 20, 2005, which claims priority to U.S.Provisional Application having Ser. No. 60/620,759 entitled “SEMI-SOFTC-CLASS IMMUNOSTIMULATORY OLIGONUCLEOTIDES” filed Oct. 20, 2004, theentire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to immunostimulatoryoligonucleotides with reduced renal inflammatory effects, compositionsthereof and methods of using the immunostimulatory oligonucleotides. Inparticular the immunostimulatory oligonucleotides are C-class semi-softoligonucleotides that are particularly effective in the treatment ofallergy and asthma, cancer and infectious disease.

BACKGROUND OF THE INVENTION

Bacterial DNA has immune stimulatory effects to activate B cells andnatural killer cells, but vertebrate DNA does not (Tokunaga, T., et al.,1988. Jpn. J. Cancer Res. 79:682-686; Tokunaga, T., et al., 1984, JNCI72:955-962; Messina, J. P., et al., 1991, J Immunol. 147:1759-1764; andreviewed in Krieg, 1998, In: Applied Oligonucleotide Technology, C. A.Stein and A. M. Krieg, (Eds.), John Wiley and Sons, Inc., New York,N.Y., pp. 431-448). It is now understood that these immune stimulatoryeffects of bacterial DNA are a result of the presence of unmethylatedCpG dinucleotides in particular base contexts (CpG motifs), which arecommon in bacterial DNA, but methylated and underrepresented invertebrate DNA (Krieg et al, 1995 Nature 374:546-549; Krieg, 1999Biochim. Biophys. Acta 93321:1-10). The immune stimulatory effects ofbacterial DNA can be mimicked with synthetic oligodeoxynucleotides (ODN)containing these CpG motifs. Such CpG ODN have highly stimulatoryeffects on human and murine leukocytes, inducing B cell proliferation;cytokine and immunoglobulin secretion; natural killer (NK) cell lyticactivity and IFN-γ secretion; and activation of dendritic cells (DCs)and other antigen presenting cells to express costimulatory moleculesand secrete cytokines, especially the Th1-like cytokines that areimportant in promoting the development of Th1-like T cell responses.These immune stimulatory effects of native phosphodiester backbone CpGODN are highly CpG specific in that the effects are dramatically reducedif the CpG motif is methylated, changed to a GpC, or otherwiseeliminated or altered (Krieg et al, 1995 Nature 374:546-549; Hartmann etal, 1999 Proc. Natl. Acad. Sci. USA 96:9305-10).

In early studies, it was thought that the immune stimulatory CpG motiffollowed the formula purine-purine-CpG-pyrimidine-pyrimidine (Krieg etal, 1995 Nature 374:546-549; Pisetsky, 1996 J. Immunol. 156:421-423;Hacker et al., 1998 EMBO J. 17:6230-6240; Lipford et al, 1998 Trends inMicrobiol. 6:496-500). However, it is now clear that mouse lymphocytesrespond quite well to phosphodiester CpG motifs that do not follow this“formula” (Yi et al., 1998 J. Immunol. 160:5898-5906) and the same istrue of human B cells and dendritic cells (Hartmann et al, 1999 Proc.Natl. Acad. Sci. USA 96:9305-10; Liang, 1996 J. Clin. Invest.98:1119-1129).

Several different classes of CpG oligonucleotides has recently beendescribed. One class is potent for activating B cells but is relativelyweak in inducing IFN-α and NK cell activation; this class has beentermed the B class. The B class CpG oligonucleotides typically are fullystabilized and include an unmethylated CpG dinucleotide within certainpreferred base contexts. See, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class of CpGoligonucleotides activates B cells and NK cells and induces IFN-α; thisclass has been termed the C-class. The C-class CpG oligonucleotides, asfirst characterized, typically are fully stabilized, include a Bclass-type sequence and a GC-rich palindrome or near-palindrome. Thisclass has been described in co-pending U.S. patent applicationUS10/224,523 filed on Aug. 19, 2002 and related PCT Patent ApplicationPCT/US02/26468 published under International Publication Number WO03/015711.

SUMMARY OF THE INVENTION

It has been discovered that immunostimulatory properties of specificC-class CpG oligonucleotides with the selective inclusion of one or morenon-stabilized linkages between certain nucleotides have significantactivity and are particularly useful in the treatment of allergy andasthma. The non-stabilized linkages are preferably natural linkages,i.e., phosphodiester linkages or phosphodiester-like linkages. Anon-stabilized linkage will typically, but not necessarily, berelatively susceptible to nuclease digestion. The immunostimulatoryoligonucleotides of the instant invention include at least onenon-stabilized linkage situated between a 5° C. and an adjacent 3′ G,wherein both the 5° C. and the 3′ G are internal nucleotides.

The immunostimulatory oligonucleotides of the instant invention areuseful for inducing a Th1-like immune response. Accordingly, theimmunostimulatory oligonucleotides of the instant invention are usefulas adjuvants for vaccination, and they are useful for treating diseasesincluding cancer, infectious disease, allergy, and asthma. They arebelieved to be of particular use in any condition calling for prolongedor repeated administration of immunostimulatory oligonucleotide for anypurpose, but are particularly useful in the treatment of asthma andallergic diseases such as allergic rhinitis.

The present invention relates in part to immunostimulatory CpGcontaining oligonucleotides. In one aspect the invention is anoligonucleotide having the formula: 5′ TC_GX₁C_G X₂N₁ X₃C_GN₂CG 3′ (SEQID NO: 26). The oligonucleotide includes at least 2 stabilizedinternucleotide linkages. “_” represents phosphodiester orphosphodiester-like internucleotide linkage. N₁ is 0-3 nucleotides inlength, N₂ is 0-9 nucleotides in length with N referring to anynucleotide. X₁, X₂, and X₃ are any nucleotide. In some embodiments X₁,X₂, and X₃ are T.

In some embodiments the oligonucleotide may comprise 5′T*C_GTC_GTN₁TC_GGCGCN₁GCCG 3′ (SEQ ID NO: 27). In one embodiment theoligonucleotide may comprise 5′ T*C_G*T*C_G*T*N₁*T*C_G*G*C*G*CN₁G*C*C*G3′ (SEQ ID NO: 27). In some embodiments N₁ is 3 or 2 nucleotides inlength. In other embodiments N₁ is 0 nucleotides in length.

The immunostimulatory oligonucleotide may comprise 5′T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′ (SEQ ID NO: 2),wherein * represents a stabilized internucleotide linkage. Optionally,when specifically stated, 5′ may refer to the free 5′ end of theoligonucleotide and 3′ may refer to the free 3′ end of theoligonucleotide.

In other embodiments the immunostimulatory oligonucleotide may comprise5′ T*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′ (SEQ ID NO: 3), wherein *represents a stabilized internucleotide linkage. Optionally, whenspecifically stated, 5′ may refer to the free 5′ end of theoligonucleotide and 3′ may refer to the free 3′ end of theoligonucleotide.

In another aspect, the immunostimulatory oligonucleotide has thefollowing formula TC_G X₁C_G X₂C_G X₃TC_GGCGC_G N₃3′ (SEQ ID NO: 28).

N₃ is 1-5 nucleotides in length with N referring to any nucleotide. Inone embodiment N₃ is 5 nucleotides. X₁, X₂, and X₃ are any nucleotide.In some embodiments X₁ and X₃ are T.

In one embodiment the oligonucleotide may comprise 5′TC_GTC_GAC_GATC_GGCGC_GCGCCG3′ (SEQ ID NO: 4), wherein theoligonucleotide includes at least 2 stabilized internucleotide linkagesand _ represents phosphodiester or phosphodiester-like internucleotidelinkage. In one embodiment the oligonucleotide may comprise 5′T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3′ (SEQ ID NO: 4).Optionally, when specifically stated, 5′ may refer to the free 5′ end ofthe oligonucleotide and 3′ may refer to the free 3′ end of theoligonucleotide.

According to another aspect of the invention an immunostimulatoryoligonucleotide having the following formula: 5′ TTC_GX₂C_GN₁X₁ _(—)GX₃C_GTT 3′ (SEQ ID NO: 24) is provided. The oligonucleotide includes atleast 2 stabilized internucleotide linkages and _ representsphosphodiester or phosphodiester-like internucleotide linkage. N₁ is 1-3nucleotides in length with N referring to any nucleotide. X₁ is apyrimidine. X₂ and X₃ are any nucleotide. In some embodiments X₂ and X₃are T.

In one embodiment the oligonucleotide may comprise 5′ TTC_GTC_GTTTX₁_(—) GTC_GTT 3′ (SEQ ID NO: 25). In another embodiment theoligonucleotide may comprise 5′ T*T*C_G*T*C_G*T*T*T*X₁ _(—) G*T*C_G*T*T3′ (SEQ ID NO: 25). In some embodiments X₁ is T or C.

The oligonucleotide may comprise 5′ T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T 3′(SEQ ID NO: 5), wherein * represents a stabilized internucleotidelinkage. Optionally, when specifically stated, 5′ may refer to the free5′ end of the oligonucleotide and 3′ may refer to the free 3′ end of theoligonucleotide.

The oligonucleotide may comprise 5′ T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T3′ (SEQ ID NO: 6), wherein * represents a stabilized internucleotidelinkage. Optionally, when specifically stated, 5′ may refer to the free5′ end of the oligonucleotide and 3′ may refer to the free 3′ end of theoligonucleotide.

In some aspects of the invention the oligonucleotide has one of thefollowing formulas TCGTCGTTCGGCGCGCCG (SEQ ID NO: 3),TCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO: 2), TCGTCGACGATCGGCGCGCGCCG (SEQ IDNO: 4), TTCGTCGTTTTGTCGTT. (SEQ ID NO: 5), or TTTCGTCGTTTCGTCGTT. (SEQID NO: 6)

In other aspects of the invention the oligonucleotide has one of thefollowing formulas TCGTCGTC, CGTCGTCG, GTCGTCGT, TCGTCGTT, CGTCGTTC,GTCGTTCG, TCGTTCGG, CGTTCGGC, GTTCGGCG, TTCGGCGC, TCGGCGCG, CGGCGCGC,GGCGCGCG, GCGCGCGC, CGCGCGCC, or GCGCGCCG.

In other aspects of the invention the oligonucleotide has one of thefollowing formulas T*C_G*T*C_G*T*C, C_G*T*C_G*T*C_G, G*T*C_G*T*C_G*T,T*C_G*T*C_G*T*T, C_G*T*C_G*T*T*C, G*T*C_G*T*T*C_G, T*C_G*T*T*C_G*G,C_G*T*T*C_G*G*C, G*T*T*C_G*G*C*G, T*T*C_G*G*C*G*C, T*C_G*G*C*G*C_G,C_G*G*C*G*C_G*C, G*G*C*G*C_G*C*G, G*C*G*C_G*C*G*C, C*G*C_G*C*G*C*C, orG*C_G*C*G*C*C*G.

In other aspects of the invention an oligonucleotide comprising:T*C_G*T*C_G*T*C, wherein * represents a stabilized internucleotidelinkage and _ represents phosphodiester or phosphodiester-likeinternucleotide linkage is provided. Optionally the oligonucleotide maybe 5′ T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C 3′ (SEQ ID NO.: 21),5′ T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C 3′ (SEQ ID NO.: 22), or 5′T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C 3′ (SEQ ID NO.: 23), wherein5′ refers to the free 5′ end of the oligonucleotide and 3′ refers to thefree 3′ end of the oligonucleotide.

In other aspects an oligonucleotide comprising: T*C_G*T*T*C_G*G,wherein * represents a stabilized internucleotide linkage and _represents phosphodiester or phosphodiester-like internucleotide linkageis provided. Optionally the oligonucleotide may be 5′C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3′ (SEQ ID NO.: 15), 5′G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3′ (SEQ ID NO.: 16), 5′T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3′ (SEQ ID NO.: 17), 5′C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3′ (SEQ ID NO.: 18), 5′G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3′ (SEQ ID NO.: 19), or 5′T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3′ (SEQ ID NO.: 20), wherein 5′ refersto the free 5′ end of the oligonucleotide and 3′ refers to the free 3′end of the oligonucleotide.

A pharmaceutical composition comprising an oligonucleotide of theinvention and a pharmaceutically acceptable carrier is provided.

In some embodiments the composition is formulated in a nebulizer or aninhaler. The inhaler may be a metered dose inhaler. Alternatively theinhaler is a powder inhaler.

In other embodiments the pharmaceutical composition may include achemotherapeutic agent. In yet other embodiments the composition mayinclude an anti-viral agent.

The pharmaceutical composition may optionally include a pharmaceuticallyacceptable carrier formulated for subcutaneous administration, oraladministration or intranasal administration.

In one embodiment the oligonucleotide is in a pharmaceutical compositionoptionally comprising a pharmaceutically acceptable carrier. In someembodiments the oligonucleotide is formulated as an aerosol.

In one embodiment the oligonucleotide further comprises an adjuvant or acytokine.

In one embodiment the oligonucleotide further comprises an antigen,wherein the oligonucleotide is a vaccine adjuvant.

In one embodiment the antigen is selected from the group consisting of:a viral antigen, a bacterial antigen, a fungal antigen, a parasiticantigen, and a tumor antigen. In one embodiment the antigen is encodedby a nucleic acid vector. In one embodiment the antigen is a peptideantigen. In one embodiment the antigen is covalently linked to theoligonucleotide or immunostimulatory nucleic acid molecule. In anotherembodiment the antigen is not covalently linked to the oligonucleotideor immunostimulatory nucleic acid molecule.

In one embodiment the phosphodiester or phosphodiester-like linkage isphosphodiester. In one embodiment the phosphodiester-like linkage isboranophosphonate or diastereomerically pure Rp phosphorothioate.

In one embodiment the stabilized backbone comprises a plurality ofinternucleotide linkages selected from the group consisting of:phosphorothioate, phosphorodithioate, methylphosphonate,methylphosphorothioate, and any combination thereof. In one embodimentthe stabilized backbone comprises a plurality of phosphorothioateinternucleotide linkages.

In one embodiment the immunostimulatory nucleic acid molecule is 4-100nucleotides long.

In other aspects the invention is a method for treating asthma byadministering to a subject having or at risk of having asthma anoligonucleotide of the invention in an effective amount to treat asthma.

In yet other aspects the invention is a method for treating allergy byadministering to a subject having or at risk of having allergy anoligonucleotide of the invention in an effective amount to treatallergy. In one embodiment the subject has allergic rhinitis. In oneembodiment the oligonucleotide is administered to a mucosal surface. Inother embodiments the oligonucleotide is administered in an aerosolformulation. Optionally the oligonucleotide is administeredintranasally.

A method for inducing cytokine production is provided according toanother aspect of the invention. The method is performed byadministering to a subject an immunostimulatory CpG oligonucleotidedescribed herein in an effective amount to induce a cytokine selectedfrom the group consisting of IL-6, IL-8, IL-12, IL-18, TNF, IFN-α,chemokines, and IFN-γ.

In another aspect the invention is a composition of the CpGimmunostimulatory oligonucleotides described herein in combination withan antigen or other therapeutic compound, such as an anti-microbialagent. The anti-microbial agent may be, for instance, an anti-viralagent, an anti-parasitic agent, an anti-bacterial agent or ananti-fungal agent.

A composition of a sustained release device including the CpGimmunostimulatory oligonucleotides described herein is providedaccording to another aspect of the invention.

The composition may optionally include a pharmaceutical carrier and/orbe formulated in a delivery device. In some embodiments the deliverydevice is selected from the group consisting of cationic lipids, cellpermeating proteins, and sustained release devices. In one embodimentthe sustained release device is a biodegradable polymer or amicroparticle.

According to another aspect of the invention a method of stimulating animmune response is provided. The method involves administering a CpGimmunostimulatory oligonucleotide to a subject in an amount effective toinduce an immune response in the subject. Preferably the CpGimmunostimulatory oligonucleotide is administered orally, locally, in asustained release device, mucosally, systemically, parenterally, orintramuscularly. When the CpG immunostimulatory oligonucleotide isadministered to the mucosal surface it may be delivered in an amounteffective for inducing a mucosal immune response or a systemic immuneresponse. In preferred embodiments the mucosal surface is selected fromthe group consisting of an oral, nasal, rectal, vaginal, and ocularsurface.

In some embodiments the method includes exposing the subject to anantigen wherein the immune response is an antigen-specific immuneresponse. In some embodiments the antigen is selected from the groupconsisting of a tumor antigen, a viral antigen, a bacterial antigen, aparasitic antigen and a peptide antigen.

CpG immunostimulatory oligonucleotides are capable of provoking a broadspectrum of immune response. For instance these CpG immunostimulatoryoligonucleotides can be used to redirect a Th2 to a Th1 immune response.CpG immunostimulatory oligonucleotides may also be used to activate animmune cell, such as a lymphocyte (e.g., B and T cells), a dendriticcell, and an NK cell. The activation can be performed in vivo, in vitro,or ex vivo, i.e., by isolating an immune cell from the subject,contacting the immune cell with an effective amount to activate theimmune cell of the CpG immunostimulatory oligonucleotide andre-administering the activated immune cell to the subject. In someembodiments the dendritic cell presents a cancer antigen. The dendriticcell can be exposed to the cancer antigen ex vivo.

The immune response produced by CpG immunostimulatory oligonucleotidesmay also result in induction of cytokine production, e.g., production ofIL-6, IL-8, IL-12, IL-18, TNF, IFN-α, chemokines, and IFN-γ.

In still another embodiment, the CpG immunostimulatory oligonucleotidesare useful for treating cancer. The CpG immunostimulatoryoligonucleotides are also useful according to other aspects of theinvention in preventing cancer (e.g., reducing a risk of developingcancer) in a subject at risk of developing a cancer. The cancer may beselected from the group consisting of biliary tract cancer, breastcancer, cervical cancer, choriocarcinoma, colon cancer, endometrialcancer, gastric cancer, intraepithelial neoplasms, lymphomas, livercancer, lung cancer (e.g. small cell and non-small cell), melanoma,neuroblastomas, oral cancer, ovarian cancer, pancreatic cancer, prostatecancer, rectal cancer, sarcomas, thyroid cancer, and renal cancer, aswell as other carcinomas and sarcomas. In some important embodiments,the cancer is selected from the group consisting of bone cancer, brainand CNS cancer, connective tissue cancer, esophageal cancer, eye cancer,Hodgkin's lymphoma, larynx cancer, oral cavity cancer, skin cancer, andtesticular cancer.

CpG immunostimulatory oligonucleotides may also be used for increasingthe responsiveness of a cancer cell to a cancer therapy (e.g., ananti-cancer therapy), optionally when the CpG immunostimulatoryoligonucleotide is administered in conjunction with an anti-cancertherapy. The anti-cancer therapy may be a chemotherapy, a vaccine (e.g.,an in vitro primed dendritic cell vaccine or a cancer antigen vaccine)or an antibody based therapy. This latter therapy may also involveadministering an antibody specific for a cell surface antigen of, forexample, a cancer cell, wherein the immune response results in antibodydependent cellular cytotoxicity (ADCC). In one embodiment, the antibodymay be selected from the group consisting of Ributaxin, Herceptin,Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN,Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-111, MDX-22, OV103,3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1,CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000,LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS,anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab andImmuRAIT-CEA.

Thus, according to some aspects of the invention, a subject havingcancer or at risk of having a cancer is administered a CpGimmunostimulatory oligonucleotide and an anti-cancer therapy. In someembodiments, the anti-cancer therapy is selected from the groupconsisting of a chemotherapeutic agent, an immunotherapeutic agent and acancer vaccine. In some embodiments the cancer medicament is taxol or acombination of carboplatin and paclitaxel.

In still another embodiment of the methods directed to preventing ortreating cancer, the subject may be further administered interferon-α.

The invention in other aspects relates to methods for preventing diseasein a subject. The method involves administering to the subject a CpGimmunostimulatory oligonucleotide on a regular basis to promote immunesystem responsiveness to prevent disease in the subject. Examples ofdiseases or conditions sought to be prevented using the prophylacticmethods of the invention include microbial infections (e.g., sexuallytransmitted diseases) and anaphylactic shock from food allergies.

In other aspects, the invention is a method for inducing an innateimmune response by administering to the subject a CpG immunostimulatoryoligonucleotide in an amount effective for activating an innate immuneresponse.

According to another aspect of the invention a method for treating orpreventing a viral or retroviral infection is provided. The methodinvolves administering to a subject having or at risk of having a viralor retroviral infection, an effective amount for treating or preventingthe viral or retroviral infection of any of the compositions of theinvention.

In some embodiments the virus is caused by a hepatitis virus e.g.,hepatitis B, hepatitis C, HIV, herpes virus, or papillomavirus.

A method for treating or preventing a bacterial infection is providedaccording to another aspect of the invention. The method involvesadministering to a subject having or at risk of having a bacterialinfection, an effective amount for treating or preventing the bacterialinfection of any of the compositions of the invention. In one embodimentthe bacterial infection is due to an intracellular bacteria.

In another aspect the invention is a method for treating or preventing aparasite infection by administering to a subject having or at risk ofhaving a parasite infection, an effective amount for treating orpreventing the parasite infection of any of the compositions of theinvention. In one embodiment the parasite infection is due to anintracellular parasite. In another embodiment the parasite infection isdue to a non-helminthic parasite.

In some embodiments the subject is a human and in other embodiments thesubject is a non-human vertebrate selected from the group consisting ofa dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat,mouse, and sheep, In another aspect the invention relates to a methodfor inducing a TH1 immune response by administering to a subject any ofthe compositions of the invention in an effective amount to produce aTH1 immune response.

In another aspect, the invention relates to a method for treatingautoimmune disease by administering to a subject having or at risk ofhaving an autoimmune disease an effective amount for treating orpreventing the autoimmune disease of any of the compositions of theinvention.

In other embodiments the oligonucleotide is delivered to the subject inan effective amount to induce cytokine expression. Optionally thecytokine is selected from the group consisting of IL-6, TNFα, IFNα, IFNγand IP-10. In other embodiments the oligonucleotide is delivered to thesubject in an effective amount to shift the immune response to a Th1biased response form a Th2 biased response.

The invention in some aspects is a method for treating airwayremodeling, comprising: administering to a subject an oligonucleotidecomprising a CG dinucleotide, in an effective amount to treat airwayremodeling in the subject. In one embodiment the subject has asthma,chronic obstructive pulmonary disease, or is a smoker. In otherembodiments the subject is free of symptoms of asthma.

Use of an oligonucleotide of the invention for stimulating an immuneresponse is also provided as an aspect of the invention.

Use of an oligonucleotide of the invention in the manufacture of amedicament of for stimulating an immune response and performing any ofthe methods of the invention is also provided.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs depicting IFN-alpha induction by human PBMCtreated with CpG ODN.

FIG. 2 is a series of graphs depicting the effects of the CpGoligodeoxynucleotide SEQ ID NO. 7 against antigen-induced increase innasal resistance in guinea pigs administered in a dose of (A) 1 mg/kgand (B) (0.03-0.3 mg/kg). Results are mean±s.e.m. ((A)=n=5-8;(B)=n=14-15). * P<0.05 compared with antigen-challenged group treatedwith test vehicle (t test).

FIG. 3 is a series of graphs depicting the effects of the CpGoligodeoxynucleotide SEQ ID NO. 7 on antigen-induced sneezing (3A) andnasal rubbing (3B) in a mouse model of allergic rhinitis. Results aremean±s.e.m. (n=10). * P<0.05 compared with vehicle-treated group(Mann-Whitney test).

FIG. 4 is a graph depicting a titration of influenza virus anddetermination of time course of infection. Cell numbers inbronchoalveolar lavage fluid. Results are mean±s.e.m. (n=5). Miceinfected with 500 EID₅₀ were sacrificed after 6 days because of weightloss.

FIG. 5 is a graph depicting the protective effects of CpG ODNs on virusload in the lung. Virus load assayed by enzyme immunoassay. Results aremean±s.e.m. (n=5-10). * P<0.05 compared with group B (Kruskal-Wallistest, Dunn's post-test). n.d.=no data.

FIG. 6 is a series of graphs depicting the protective effects of CpGODNs on virus-induced airways inflammation measuring total leukocytes(6A), total neutrophils (6B) and total mononuclear cells (6C). Cellnumbers in bronchoalveolar lavage fluid. Results are mean±s.e.m.(n=10). * P<0.05 compared with group B (Kruskal-Wallis test, Dunn'spost-test).

FIG. 7 is a series of graphs depicting the protective effects of CpGODNs on cell numbers in antigen-induced airways inflammation ineosinophils (7A) and neutrophils (7B). Results are mean±s.e.m.(n=10-14). * P<0.05 compared with antigen challenged, vehicle-treatedgroup (Kruskal-Wallis multiple comparison test, Dunn's post test).

FIG. 8 is a series of graphs depicting the protective effects of CpGODNs on CD3⁺ (8A) and CD3⁺CD4⁺ (8B) cell numbers in antigen-inducedairways inflammation. Results are mean±s.e.m. (n=8). * P<0.05 comparedwith antigen challenged, vehicle-treated group (Kruskal-Wallis multiplecomparison test, Dunn's post test).

FIG. 9 is a series of graphs depicting cell numbers in bronchoalveolarlavage fluid 8 hours after dosing with 0.1 mg/kg ODN (9A); 15 hoursafter dosing with 0.1 mg/kg ODN (9B); 8 hours after dosing with 1 mg/kgODN (9C); and 15 hours after dosing with 1 mg/kg ODN (9D). Results aremean±s.e.m (n=10)

FIG. 10 is a series of graphs depicting concentrations of IFN alpha inbronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg/kg ODN(10A); 15 hours after dosing with 0.1 mg/kg ODN (10B); 8 hours afterdosing with 1 mg/kg ODN (10C); and 15 hours after dosing with 1 mg/kgODN (10D).

FIG. 11 is a series of graphs depicting concentrations of IFN gamma inbronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg/kg ODN(11A); 15 hours after dosing with 0.1 mg/kg ODN (11B); 8 hours afterdosing with 1 mg/kg ODN (11C); and 15 hours after dosing with 1 mg/kgODN (11D).

FIG. 12 is a series of graphs depicting concentrations of IP-10 inbronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg/kg ODN(12A); 15 hours after dosing with 0.1 mg/kg ODN (12B); 8 hours afterdosing with 1 mg/kg ODN (12C); and 15 hours after dosing with 1 mg/kgODN (12D).

FIG. 13 is a series of graphs depicting concentrations of IL-12p40 inbronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg/kg ODN(13A); 15 hours after dosing with 0.1 mg/kg ODN (13B); 8 hours afterdosing with 1 mg/kg ODN (13C); and 15 hours after dosing with 1 mg/kgODN (13D).

FIG. 14 is a series of graphs depicting concentrations of IL-6 inbronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg/kg ODN(14A); 15 hours after dosing with 0.1 mg/kg ODN (14B); 8 hours afterdosing with 1 mg/kg ODN (14C); and 15 hours after dosing with 1 mg/kgODN (14D).

FIG. 15 is a series of graphs depicting concentrations of TNFalpha inbronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg/kg ODN(15A); 15 hours after dosing with 0.1 mg/kg ODN (15B); 8 hours afterdosing with 1 mg/kg ODN (15C); and 15 hours after dosing with 1 mg/kgODN (15D).

FIG. 16 is a series of graphs depicting concentrations of IFN gamma inserum 8 hours after dosing with 0.1 mg/kg ODN (16A); 15 hours afterdosing with 0.1 mg/kg ODN (16B); 8 hours after dosing with 1 mg/kg ODN(16C); and 15 hours after dosing with 1 mg/kg ODN (16D).

FIG. 17 is a series of graphs depicting concentrations of IL-6 in serum8 hours after dosing with 0.1 mg/kg ODN (17A); 15 hours after dosingwith 0.1 mg/kg ODN (17B); 8 hours after dosing with 1 mg/kg ODN (17C);and 15 hours after dosing with 1 mg/kg ODN (17D).

FIG. 18 is a series of graphs depicting concentrations of TNF alpha inserum 8 hours after dosing with 0.1 mg/kg ODN (18A); 15 hours afterdosing with 0.1 mg/kg ODN (18B); 8 hours after dosing with 1 mg/kg ODN(18C); and 15 hours after dosing with 1 mg/kg ODN (18D).

FIG. 19 is a series of graphs depicting the effects of CpGoligodeoxynucleotides ODN SEQ ID NO: 2 and ODN SEQ ID NO: 7 onantigen-induced IgE (19A) and IgG2a (19B) production in the mouse.Results are mean±s.e.m. (n=10). * P<0.05 compared with antigensensitized, vehicle-treated group (Kruskal-Wallis test with Dunn's posttest).

FIG. 20 is a series of graphs depicting Effects of ODN SEQ ID NO: 2against influenza-induced exacerbation of allergic airway inflammationin mice, depicting total leukocytes (20A), eosinophils (20B),neutrophils (20C), mononuclear cells (20D), and body weight (20E).

FIG. 21 is a guinea pig AHR Protocol used herein.

FIG. 22 is a set of graphs depicting the effect of SEQ ID NO:7 on Airwayresistance and lung compliance. For each animal, a dose-response curvewas obtained, and airway reactivity was quantified as the area under thecurve. Guinea pigs were given intranasally either carrier (saline), OVAalone, or concentrations of SEQ ID NO:7 of 10 μl/kg, 30 μl/kg, 100μl/kg, or 300 μl/kg, i.t. The data demonstrate that SEQ ID NO:7 caused adose-dependent reduction in AUC-resistance. FIGS. 22A and 22B depicthistamine release as a function of increased airway resistance (22A) ordecrease in lung compliance (22B). FIGS. 22C and 22D are bar graphsdepicting the increase in airway resistance (22C) or decrease in lungcompliance (22D) in response to treatment with saline, OVA or SEQ ID NO7 at the indicated dosages.

FIG. 23 is a Summary of Statistical Analysis used herein (FIG. 22) forairway resistance (23A) and lung compliance (23B).

FIG. 24 is a set of graphs depicting the effect of SEQ ID NO:2 on Airwayresistance and lung compliance. For each animal, a dose-response curvewas obtained, and airway reactivity was quantified as the area under thecurve. Guinea pigs were given intranasally either carrier (saline), OVAalone, or concentrations of SEQ ID NO:2 of 10 μl/kg, 30 μl/kg, 100 μl/kgor 300 μl/kg, i.t. The data demonstrate that SEQ ID NO:2 caused adose-dependent reduction in AUC-resistance. FIGS. 24A and 24B depicthistamine release as a function of increased airway resistance (24A) ordecrease in lung compliance (24B). FIGS. 24C and 24D are bar graphsdepicting the increase in airway resistance (24C) or decrease in lungcompliance (24D) in response to treatment with saline, OVA or SEQ ID NO2 at the indicated dosages.

FIG. 25 is a Summary of Statistical Analysis used herein (FIG. 24) forairway resistance (25A) and lung compliance (25B)

FIG. 26 is a summary of the graphs in FIGS. 22 and 24. FIGS. 26A and 26Bcorrespond to FIGS. 22C and 22D. FIGS. 26C and 26D correspond to FIGS.24C and 24D.

FIG. 27 is a set of graphs depicting levels of IL-10 μg/ml) secretedfrom human PBMC (3 donors) following exposure of these cells to theoligonucleotides listed by SEQ ID No. along the bottom X-axis of thegraph (data points from the 3 donors are depicted by a ▴, ▪ and x) for48 hours. The test oligonucleotides shown in FIG. 27 include SEQ ID NOs:10, 9, 13, 14, 1, and 2. The concentration of oligonucleotide used toproduce a particular data point is depicted along the X-axis (μM).Supernatants were harvested and IL-10 measured by ELISA. Given are themean cytokine amounts of all donors.

FIG. 28 is a set of graphs depicting levels of TNF-alpha (28A),interferon-gamma (28B), and IL-6 (28C) (μM) secreted from human PBMCfollowing exposure of these cells to the oligonucleotides listed by SEQID NO. in the key of the graph. Each data point is the calculated meancytokine value of three donors. The PBMC were incubated with theindicated ODN concentrations. Supernatants were harvested and cytokinesmeasured by ELISA. Given are the calculated mean cytokine amounts of alldonors.

FIG. 29 is a set of graphs depicting levels of TNF-alpha (pg/ml)secreted from human PBMC following exposure of these cells to theoligonucleotides for 16 hours listed by the SEQ ID NO along the bottomX-axis of the graph. The oligonucleotides shown in FIG. 29 include SEQID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM). The data shown represents the values of threedonors. Below the SEQ ID NOs is a designation referring to the class ofODN. Css=C-class semi soft, C=C-class, B=B-class, non-CpG=an ODN withoutan unmethylated CpG. Supernatants were harvested and IL-6 measured byELISA. Given are the mean cytokine amounts of all donors.

FIG. 30 is a set of graphs depicting levels of IL-6 (pg/ml) secretedfrom human PBMC following exposure of these cells to theoligonucleotides for 24 hours listed by the SEQ ID NO along the bottomX-axis of the graph. The oligonucleotides shown in FIG. 30 include SEQID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM). The data shown represents the values of threedonors. Below the SEQ ID NOs is a designation referring to the class ofODN. Css=C-class semi soft, C=C-class, B=B-class, non-CpG=an ODN withoutan unmethylated CpG.

FIG. 31 is a set of graphs depicting levels of IFN-gamma (pg/ml)secreted from human PBMC following exposure of these cells to theoligonucleotides for 48 hours listed by the SEQ ID NO along the bottomX-axis of the graph. The oligonucleotides shown in FIG. 31 include SEQID NO. 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM). The data shown represents the values of threedonors, Below the SEQ ID NOs is a designation referring to the class ofODN. Css=C-class semi soft, C=C-class, B=B-class, non-CpG=an ODN withoutan unmethylated CpG. Supernatants were harvested and IFN-gamma measuredby ELISA. Given are the mean cytokine amounts of all donors.

FIG. 32 is a set of graphs depicting levels of CD69 expression (MFI) onNK cells as an indicator of NK cell activation (32A) and CD80 (32B) andCD86 (32C) expression on B cells following exposure of these cells tothe oligonucleotides for 24 or 48 hours listed by SEQ ID NO. in the keyof the graph. Each data point is the mean fluorescence intensity ofthree donors. The cells were incubated with the indicated ODNconcentrations for 24, or 48 hours. Cells were stained and analyzed byflow cytometry.

FIG. 33 is a set of graphs depicting levels of levels of CD69 expressionon NK cells as an indicator of NK cell activation following exposure ofthese cells to the oligonucleotides for 24 hours listed by the SEQ ID NOalong the bottom X-axis of the graph. The oligonucleotides shown in FIG.33 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM). The data shown represents the valuesof three donors. Below the SEQ ID NOs is a designation referring to theclass of ODN. Css=C-class semi soft, C=C-class, B=B-class, non-CpG=anODN without an unmethylated CpG. The cells were stained with antibodiesto CD3, CD56, and CD69 and analyzed by flow cytometry. The datapresented is the mean fluorescence intensity.

FIG. 34 is a set of graphs depicting CD86 expression on human PBMCfollowing exposure of these cells to the oligonuelcotides for 48 hourslisted by the SEQ ID NO along the bottom X-axis of the graph. Theoligonucleotides shown in FIG. 34 include SEQ ID NO: 9, 13, 10, 14, 2,1, 11, 12, and 3. The concentration of oligonucleotide used to produce aparticular data point is depicted along the X-axis (μM). The data shownrepresents the values of three donors. Below the SEQ ID NOs is adesignation referring to the class of ODN. Css=C-class semi soft,C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG. Thecells were stained with antibodies to CD86, CD80, CD19, and CD14 andanalyzed by flow cytometry. The data presented is the mean fluorescenceintensity.

FIG. 35 is a set of graphs depicting CD86 expression on human PBMCfollowing exposure of these cells to the oligonucleotides for 48 hourslisted by the SEQ ID NO along the bottom X-axis of the graph. Theoligonucleotides shown in FIG. 35 include SEQ ID NO: 9, 13, 10, 14, 2,1, 11, 12, and 3. The concentration of oligonucleotide used to produce aparticular data point is depicted along the X-axis (μM). The data shownrepresents the values of three donors. Below the SEQ ID NOs is adesignation referring to the class of ODN. Css=C-class semi soft,C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG. Thecells were stained with antibodies to CD86, CD80, CD19, and CD14 andanalyzed by flow cytometry. The data presented is the mean fluorescenceintensity.

FIG. 36 is a set of graphs depicting levels of CD86 expression onplasmacytoid dendritic cells (36A) and CDS0 (36B) and CD86 (36C)expression on monocytes following exposure of these cells to theoligonucleotides listed by SEQ ID NO. in the key of the graph. Each datapoint is the a calculated mean fluorescence intensity of three donors.The cells were incubated with the indicated ODN concentrations for 48hours. Cells were stained and analyzed by flow cytometry.

FIG. 37 is a set of graphs depicting levels of CD86 expression onmonocytes following exposure of the cells to the oligonucleotides for 48hours listed by the SEQ ID NO along the bottom X-axis of the graph. Theoligonucleotides shown in FIG. 37 include SEQ ID NO: 9, 13, 10, 14, 2,1, 11, 12, and 3. The concentration of oligonucleotide used to produce aparticular data point is depicted along the X-axis (μM). The data shownrepresents the values of three donors. Below the SEQ ID NOs is adesignation referring to the class of ODN. Css=C-class semi soft,C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG. Thecells were stained with antibodies to CD86, CD80, CD19, and CD14 andanalyzed by flow cytometry. The data presented is the mean fluorescenceintensity.

FIG. 38 is a set of graphs depicting CD80 expression on monocytesfollowing exposure of these cells to the oligonucleotides for 48 hourslisted by the SEQ ID NO along the bottom X-axis of the graph. Theoligonucleotides shown in FIG. 38 include SEQ ID NO: 9, 13, 10, 14, 2,1, 11, 12, and 3. The concentration of oligonucleotide used to produce aparticular data point is depicted along the X-axis (μM). The data shownrepresents the values of three donors. Below the SEQ ID NOs is adesignation referring to the class of ODN. Css=C-class semi soft,C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG. Thecells were stained with antibodies to CD86, CD80, CD19, and CD14 andanalyzed by flow cytometry. The data presented is the mean fluorescenceintensity.

FIG. 39 is a set of graphs depicting CD80 expression on plasmacytoiddendritic cells following exposure of these cells to theoligonucleotides for 48 hours listed by the SEQ ID NO along the bottomX-axis of the graph. The oligonucleotides shown in FIG. 37 include SEQID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM). The data shown represents the values of threedonors. Below the SEQ ID NOs is a designation referring to the class ofODN. Css=C-class semi soft, C=C-class, B=B-class, non-CpG=an ODN withoutan unmethylated CpG. The cells were stained with antibodies to CD86,CD11c, CD123, and HLA-DR and analyzed by flow cytometry. The datapresented is the mean fluorescence intensity.

FIG. 40 is a set of graphs depicting levels of expression ofintracellular IP-10 in B cells (40B) and monocytes (40A) followingexposure of these cells to the oligonucleotides for 24 hours listed bySEQ ID NO. in the key of the graph. Each data point is the a calculatedmean fluorescence intensity of three donors. The cells were incubatedwith the indicated ODN concentrations for 24 hours. Cells were stainedand analyzed by flow cytometry.

FIG. 41 is a set of graphs depicting levels of expression ofintracellular IP-10 in monocytes following exposure of the cells to theoligonucleotides for 24 hours listed by the SEQ ID NO along the bottomX-axis of the graph. The oligonucleotides shown in FIG. 41 include SEQID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM). The data shown represents the values of threedonors. Below the SEQ ID NOs is a designation referring to the class ofODN. Css=C-class semi soft, C=C-class, B=B-class, non-CpG=an ODN withoutan unmethylated CpG. The cells were stained with antibodies to CD14,CD19, and IP-10 and analyzed by flow cytometry. The data presented isthe mean fluorescence intensity.

FIG. 42 is a set of graphs depicting levels of expression ofintracellular IP-10 in B cells following exposure of these cells to theoligonucleotides for 24 hours listed by the SEQ ID NO along the bottomX-axis of the graph. The oligonucleotides shown in FIG. 42 include SEQID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM). The data shown represents the values of threedonors. Below the SEQ ID NOs is a designation referring to the class ofODN. Css=C-class semi soft, C=C-class, B=B-class, non-CpG=an ODN withoutan unmethylated CpG. The cells were stained with antibodies to CD14,CD19, and IP-10 and analyzed by flow cytometry. The data presented isthe mean fluorescence intensity.

FIG. 43 is a set of graphs depicting a comparison of the abilities ofSEQ ID NO.: 2 and fragments thereof (SEQ ID NO 15-17) to induce cytokinesecretion from mouse splenocytes. The cytokines analyzed include IFNα(43A), IFNγ (43B), IP-10 (43C), IL-6 (43D), IL-10 (43E), and TNFα (43F).

FIG. 44 is a set of graphs depicting a comparison of the abilities ofSEQ ID NO.: 2 and fragments thereof (SEQ ID NO 18-20) to induce cytokinesecretion from mouse splenocytes. The cytokines analyzed include IFNα(44A), IFNγ (44B), IP-10 (44C), IL-6 (44D), IL-10 (44E), and TNFα (44F).

FIG. 45 is a set of graphs depicting a comparison of the abilities ofSEQ ID NO.: 2 and fragments thereof (SEQ ID NO 21-23) to induce cytokinesecretion from mouse splenocytes. The cytokines analyzed include IFNα(45A), IFNγ (45B), IP-10 (45C), IL-6 (45D), IL-10 (45E), and TNFα (45F).

DETAILED DESCRIPTION

A sub-set of C-class semi-soft immunostimulatory oligonucleotides areprovided according to the invention. The immunostimulatoryoligonucleotides of the invention described herein, in some embodimentshave improved properties including similar or enhanced potency, reducedsystemic exposure to the kidney, liver and spleen, and may have reducedreactogenicity at injection sites. Although applicant is not bound by amechanism, it is believed that these improved properties are associatedwith the strategic placement within the immunostimulatoryoligonucleotides of phosphodiester or phosphodiester-like“internucleotide linkages”. The term “internucleotide linkage” as usedherein refers to the covalent backbone linkage joining two adjacentnucleotides in a nucleic acid molecule. The covalent backbone linkagewill typically be a modified or unmodified phosphate linkage, but othermodifications are possible. Thus a linear oligonucleotide that is nnucleotides long has a total of n−1 internucleotide linkages. Thesecovalent backbone linkages can be modified or unmodified in theimmunostimulatory oligonucleotides according to the teachings of theinvention.

In particular, phosphodiester or phosphodiester-like internucleotidelinkages involve “internal dinucleotides”. An internal dinucleotide ingeneral shall mean any pair of adjacent nucleotides connected by aninternucleotide linkage, in which neither nucleotide in the pair ofnucleotides is a terminal nucleotide, i.e., neither nucleotide in thepair of nucleotides is a nucleotide defining the 5′ or 3′ end of theoligonucleotide. Thus a linear oligonucleotide that is n nucleotideslong has a total of n−1 dinucleotides and only n−3 internaldinucleotides. Each internucleotide linkage in an internal dinucleotideis an internal internucleotide linkage. Thus a linear oligonucleotidethat is n nucleotides long has a total of n−1 internucleotide linkagesand only n−3 internal internucleotide linkages. The strategically placedphosphodiester or phosphodiester-like internucleotide linkages,therefore, refer to phosphodiester or phosphodiester-likeinternucleotide linkages positioned between any pair of nucleotides inthe nucleic acid sequence. In some embodiments the phosphodiester orphosphodiester-like internucleotide linkages are not positioned betweeneither pair of nucleotides closest to the 5′ or 3′ end.

The invention is based at least in some aspects on the surprisingdiscovery that the specific C-class semi-soft oligonucleotides describedherein have important immunostimulatory activity and are preferablyuseful in the treatment of allergy and asthma. These molecules have atleast the same or in many cases possess greater immunostimulatoryactivity, in many instances, than corresponding fully stabilizedimmunostimulatory oligonucleotides having the same nucleotide sequence.

A semi-soft oligonucleotide is an immunostimulatory oligonucleotidehaving a partially stabilized backbone, in which phosphodiester orphosphodiester-like internucleotide linkages occur only within at leastone internal pyrimidine-purine (YZ, preferably CG) dinucleotide.Semi-soft oligonucleotides generally possess increased immunostimulatorypotency relative to corresponding fully stabilized immunostimulatoryoligonucleotides. Due to the greater potency of semi-softoligonucleotides, semi-soft oligonucleotides may be used at lowereffective concentrations and have lower effective doses thanconventional fully stabilized immunostimulatory oligonucleotides inorder to achieve a desired biological effect.

Whereas fully stabilized immunostimulatory oligonucleotides can exhibitdose-response maxima, semi-soft oligonucleotides of the instantinvention appear to have monotonically increasing dose-response curves(as assayed by TLR9 stimulation) extending into higher concentrationsbeyond the optimal concentration for corresponding fully stabilizedimmunostimulatory oligonucleotides. Thus it is believed that semi-softoligonucleotides of the instant invention may induce greaterimmunostimulation than fully stabilized immunostimulatoryoligonucleotides.

Whereas fully stabilized immunostimulatory oligonucleotides less than 20nucleotides long can have modest immunostimulatory activity comparedwith longer (e.g., 24 nucleotides long) fully stabilizedoligonucleotides, semi-soft oligonucleotides as short as 16 nucleotideslong have been discovered to have immunostimulatory activity at leastequal to immunostimulatory activity of fully stabilized oligonucleotidesover 20 nucleotides long.

In some instances where a 6-mer phosphorothioate oligonucleotideappeared to lack immunostimulatory activity, substitution of even onephosphodiester internal CG internucleotide linkage for aphosphorothioate linkage was found to yield a corresponding 6-mer withimmunostimulatory activity.

Thus the size (i.e., the number of nucleotide residues along the lengthof the oligonucleotide) of the immunostimulatory oligonucleotide mayalso contribute to the stimulatory activity of the oligonucleotide. Forfacilitating uptake into cells immunostimulatory oligonucleotides mayhave a minimum length of 6 nucleotide residues. Oligonucleotides of anysize greater than 6 nucleotides (even many kb long) are capable ofinducing an immune response according to the invention if sufficientimmunostimulatory motifs are present, since larger nucleic acids aredegraded inside of cells. It is believed by the instant inventors thatsemi-soft oligonucleotides as short as 4 nucleotides can also beimmunostimulatory if they can be delivered to the interior of the cell.In certain preferred embodiments according to the instant invention, theimmunostimulatory oligonucleotides are between 4 and 100 nucleotideslong. In typical embodiments the immunostimulatory oligonucleotides arebetween 6 and 40 or 10 and 40 nucleotides long. In certain preferredembodiments according to the instant invention, the immunostimulatoryoligonucleotides are between 6 and 19 or 6 and 24 nucleotides long.

It is also believed that the foregoing properties of semi-softoligonucleotides generally increase with increasing “dose” ofphosphodiester or phosphodiester-like internucleotide linkages involvinginternal CG dinucleotides. Thus it is believed, for example, thatgenerally for a given oligonucleotide sequence with five internal CGdinucleotides, an oligonucleotide with five internal phosphodiester orphosphodiester-like CG internucleotide linkages is moreimmunostimulatory than an oligonucleotide with four internalphosphodiester or phosphodiester-like CG internucleotide linkages, whichin turn is more immunostimulatory than an oligonucleotide with threeinternal phosphodiester or phosphodiester-like CG internucleotidelinkages, which in turn is more immunostimulatory than anoligonucleotide with two internal phosphodiester or phosphodiester-likeCG internucleotide linkages, which in turn is more immunostimulatorythan an oligonucleotide with one internal phosphodiester orphosphodiester-like CG internucleotide linkage. Importantly, inclusionof even one internal phosphodiester or phosphodiester-like CGinternucleotide linkage is believed to be advantageous over no internalphosphodiester or phosphodiester-like CG internucleotide linkage. Inaddition to the number of phosphodiester or phosphodiester-likeinternucleotide linkages, the position along the length of theoligonucleotide can also affect potency.

The immunostimulatory oligonucleotides of the present invention aregenerally protected from rapid degradation in the serum. Theimmunostimulatory oligonucleotides of the present invention are alsogenerally protected from rapid degradation in most tissues, with theexception of particular tissues with specific or excessive nucleaseactivity that are capable of degrading the immunostimulatoryoligonucleotides. This results in the reduction of immunostimulatoryoligonucleotides in those particular tissues, the accumulation of whichcould otherwise lead to undesirable effects from long-term therapyutilizing degradation-resistant oligonucleotides. The oligonucleotidesof the instant invention will generally include, in addition to thephosphodiester or phosphodiester-like internucleotide linkages atpreferred internal positions, 5′ and 3′ ends that are resistant todegradation. Such degradation-resistant ends can involve any suitablemodification that results in an increased resistance against exonucleasedigestion over corresponding unmodified ends. For instance, the 5′ and3′ ends can be stabilized by the inclusion there of at least onephosphate modification of the backbone. In a preferred embodiment, theat least one phosphate modification of the backbone at each end isindependently a phosphorothioate, phosphorodithioate, methylphosphonate,or methylphosphorothioate internucleotide linkage. In anotherembodiment, the degradation-resistant end includes one or morenucleotide units connected by peptide or amide linkages at the 3′ end.Yet other stabilized ends, including but not limited to those describedfurther below, are meant to be encompassed by the invention.

As described above, the oligonucleotides of the instant inventioninclude phosphodiester or phosphodiester-like linkages within andoptionally adjacent to internal CG dinucleotides. Such CG dinucleotidesare frequently part of immunostimulatory motifs. It is not necessary,however, that an oligonucleotide contain phosphodiester orphosphodiester-like linkages within every immunostimulatory motif.Additional phosphodiester or phosphodiester-like linkages may also bemaintained for even more rapid renal digestion of these otherwise“stabilized oligonucleotides”.

A phosphodiester internucleotide linkage is the type of linkagecharacteristic of nucleic acids found in nature. The phosphodiesterinternucleotide linkage includes a phosphorus atom flanked by twobridging oxygen atoms and bound also by two additional oxygen atoms, onecharged and the other uncharged. Phosphodiester internucleotide linkageis particularly preferred when it is important to reduce the tissuehalf-life of the oligonucleotide.

A phosphodiester-like internucleotide linkage is a phosphorus-containingbridging group that is chemically and/or diastereomerically similar tophosphodiester. Measures of similarity to phosphodiester includesusceptibility to nuclease digestion and ability to activate RNAse H.Thus for example phosphodiester, but not phosphorothioate,oligonucleotides are susceptible to nuclease digestion, while bothphosphodiester and phosphorothioate oligonucleotides activate RNAse H.In a preferred embodiment the phosphodiester-like internucleotidelinkage is boranophosphate (or equivalently, boranophosphonate) linkage.U.S. Pat. No. 5,177,198; U.S. Pat. No. 5,859,231; U.S. Pat. No.6,160,109; U.S. Pat. No. 6,207,819; Sergueev et al., (1998) J Am ChemSoc 120:9417-27. In another preferred embodiment the phosphodiester-likeinternucleotide linkage is diasteriomerically pure Rp phosphorothioate.It is believed that diasteriomerically pure Rp phosphorothioate is moresusceptible to nuclease digestion and is better at activating RNAse Hthan mixed or diastereomerically pure Sp phosphorothioate. It is to benoted that for purposes of the instant invention, the term“phosphodiester-like internucleotide linkage” specifically excludesphosphorodithioate and methylphosphonate internucleotide linkages.

The immunostimulatory oligonucleotide molecules of the instant inventionhave chimeric backbone. For purposes of the instant invention, achimeric backbone refers to a partially stabilized backbone, wherein atleast one internucleotide linkage is phosphodiester orphosphodiester-like, and wherein at least one other internucleotidelinkage is a stabilized internucleotide linkage, wherein the at leastone phosphodiester or phosphodiester-like linkage and the at least onestabilized linkage are different. Since boranophosphonate linkages havebeen reported to be stabilized relative to phosphodiester linkages, forpurposes of the chimeric nature of the backbone, boranophosphonatelinkages can be classified either as phosphodiester-like or asstabilized, depending on the context. For example, a chimeric backboneaccording to the instant invention could in one embodiment include atleast one phosphodiester (phosphodiester or phosphodiester-like) linkageand at least one boranophosphonate (stabilized) linkage. In anotherembodiment a chimeric backbone according to the instant invention couldinclude boranophosphonate (phosphodiester or phosphodiester-like) andphosphorothioate (stabilized) linkages. A “stabilized internucleotidelinkage” shall mean an internucleotide linkage that is relativelyresistant to in vivo degradation (e.g., via an exo- or endo-nuclease),compared to a phosphodiester internucleotide linkage. Preferredstabilized internucleotide linkages include, without limitation,phosphorothioate, phosphorodithioate, methylphosphonate, andmethylphosphorothioate. Other stabilized internucleotide linkagesinclude, without limitation: peptide, alkyl, dephospho, and others asdescribed above.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described. Uhlmann E et al. (1990) Chem Rev 90:544; GoodchildJ (1990) Bioconjugate Chem 1:165. Methods for preparing chimericoligonucleotides are also known. For instance patents issued to Uhlmannet al have described such techniques.

Mixed backbone modified ODN may be synthesized using a commerciallyavailable DNA synthesizer and standard phosphoramidite chemistry. (F. E.Eckstein, “Oligonucleotides and Analogues—A Practical Approach” IRLPress. Oxford, UK, 1991, and M. D. Matteucci and M. H. Caruthers,Tetrahedron Lett. 21, 719 (1980)) After coupling, PS linkages areintroduced by sulfurization using the Beaucage reagent (R. P. Iyer, W.Egan, J. B. Regan and S. L. Beaucage, J. Am. Chem. Soc. 112, 1253(1990)) (0.075 M in acetonitrile) or phenyl acetyl disulfide (PADS)followed by capping with acetic anhydride, 2,6-lutidine intetrahydrofurane (1:1:8; v:v:v) and N-methylimidazole (16% intetrahydrofurane). This capping step is performed after thesulfurization reaction to minimize formation of undesired phosphodiester(PO) linkages at positions where a phosphorothioate linkage should belocated. In the case of the introduction of a phosphodiester linkage,e.g. at a CpG dinucleotide, the intermediate phosphorous-III is oxidizedby treatment with a solution of iodine in water/pyridine. After cleavagefrom the solid support and final deprotection by treatment withconcentrated ammonia (15 hrs at 50° C.), the ODN are analyzed by HPLC ona Gen-Pak Fax column (Millipore-Waters) using a NaCl-gradient (e.g.buffer A: 10 mM NaH₂PO₄ in acetonitrile/water=1:4/v:v pH 6.8; buffer B:10 mM NaH₂PO₄, 1.5 M NaCl in acetonitrile/water=1:4/v:v; 5 to 60% B in30 minutes at 1 ml/min) or by capillary gel electrophoresis. The ODN canbe purified by HPLC or by FPLC on a Source High Performance column(Amersham Pharmacia). HPLC-homogeneous fractions are combined anddesalted via a C18 column or by ultrafiltration. The ODN was analyzed byMALDI-TOF mass spectrometry to confirm the calculated mass.

The oligonucleotides of the invention can also include othermodifications. These include nonionic DNA analogs, such as alkyl- andaryl-phosphates (in which the charged phosphonate oxygen is replaced byan alkyl or aryl group), phosphodiester and alkylphosphotriesters, inwhich the charged oxygen moiety is alkylated. Oligonucleotides whichcontain diol, such as tetraethyleneglycol or hexaethyleneglycol, ateither or both termini have also been shown to be substantiallyresistant to nuclease degradation.

The oligonucleotides of the present invention are nucleic acids thatcontain specific sequences found to elicit an immune response. Thesespecific sequences that elicit an immune response are referred to as“immunostimulatory motifs”, and the oligonucleotides that containimmunostimulatory motifs are referred to as “immunostimulatory nucleicacid molecules” and, equivalently, “immunostimulatory nucleic acids” or“immunostimulatory oligonucleotides”. The immunostimulatoryoligonucleotides of the invention thus include at least oneimmunostimulatory motif. In a preferred embodiment the immunostimulatorymotif is an “internal immunostimulatory motif”. The term “internalimmunostimulatory motif” refers to the position of the motif sequencewithin a longer nucleic acid sequence, which is longer in length thanthe motif sequence by at least one nucleotide linked to both the 5′ and3′ ends of the immunostimulatory motif sequence.

The immunostimulatory oligonucleotides include immunostimulatory motifswhich are “CpG dinucleotides”. A CpG dinucleotide can be methylated orunmethylated. An immunostimulatory oligonucleotide containing at leastone unmethylated CpG dinucleotide is a nucleic acid molecule whichcontains an unmethylated cytosine-guanine dinucleotide sequence (i.e.,an unmethylated 5′ cytidine followed by 3′ guanosine and linked by aphosphate bond) and which activates the immune system; such animmunostimulatory oligonucleotide is a CpG oligonucleotide. CpGoligonucleotides have been described in a number of issued patents,published patent applications, and other publications, including U.S.Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and6,339,068. An immunostimulatory oligonucleotide containing at least onemethylated CpG dinucleotide is an oligonucleotide which contains amethylated cytosine-guanine dinucleotide sequence (i.e., a methylated 5′cytidine followed by a 3′ guanosine and linked by a phosphate bond) andwhich activates the immune system.

It has recently been described that there are different classes of CpGoligonucleotides. One class is potent for activating B cells but isrelatively weak in inducing IFN-α and NK cell activation; this class hasbeen termed the B class. The B class CpG oligonucleotides typically arefully stabilized and include an unmethylated CpG dinucleotide withincertain preferred base contexts. See, e.g., U.S. Pat. Nos. 6,194,388;6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another classis potent for inducing IFN-α and NK cell activation but is relativelyweak at stimulating B cells; this class has been termed the A class. TheA class CpG oligonucleotides typically have stabilized poly-G sequencesat 5′ and 3′ ends and a central palindromic phosphodiester CpGdinucleotide-containing sequence of at least 6 nucleotides. See, forexample, published patent application PCT/US00/26527 (WO 01/22990).

Yet another class of CpG oligonucleotides activates B cells and NK cellsand induces IFN-α; this class has been termed the C-class. The C-classCpG oligonucleotides, as first characterized, typically are fullystabilized, include a B class-type sequence and a GC-rich palindrome ornear-palindrome. This class has been described in co-pending U.S. patentapplication Ser. No. 10/224,523, filed Aug. 19, 2002 and published asUS2003/0148976 and US10/978,283 filed on Oct. 29, 2004, with a relatedPCT application published as WO2005/042018 the entire contents of whichare incorporated herein by reference.

C-class oligonucleotides are also referred to as type C CpG ODN. Incertain embodiments the C CpG ODN involve a combination of motifswherein one motif is a CG-rich palindrome or a neutralizing motif, andanother motif is a stimulatory motif; e.g., a CpG motif or the sequenceTCGTCG.

The C CpG ODN may have the formula: 5′ X₁DCGHX₂ 3′. X₁ and X₂ areindependently any sequence 0 to 10 nucleotides long. D is a nucleotideother than C. C is cytosine. G is guanine. H is a nucleotide other thanG. The nucleic acid sequence also includes a nucleic acid sequenceselected from the group consisting of P and N positioned immediately 5′to X₁ or immediately 3′ to X₂ N is a B-cell neutralizing sequence whichbegins with a CGG trinucleotide and is at least 10 nucleotides long. Pis a GC-rich palindrome containing sequence at least 10 nucleotideslong.

In some embodiments the immunostimulatory nucleic acid is 5′ NX₁DCGHX₂3′, 5′ X₁DCGHX₂N 3′, 51 PX₁DCGHX₂ 3′, 5<X₁DCGHX₂P 3′, 5′ X₁DCGHX₂PX₃ 3′,5′ X₁DCGHPX₃ 3′, 5′ DCGHX₂PX₃ 3′, 5′ TCGHX₂PX₃ 3′, or 5′ DCGHPX₃ 3′. X₃is any sequence 0 to 10 nucleotides long. In other embodiments theimmunostimulatory nucleic acid is 5′ DCGHP 3′.

Optionally D and/or H are thymine (T).

In other embodiments H is T and X₂ is CG, CGT, CGTT, CGTTT, or CGTTTT.

H is T and X₂ is CG or CGTTTT according to other embodiments.

According to other embodiments C is unmethylated.

N includes at least four CG dinucleotides and no more than two CCGtrinucleotides in some embodiments.

Optionally P includes at least one Inosine.

The nucleic acid may also include a poly-T sequence at the 5′ end or the3′ end.

Alternatively the C CpG ODN may have the formula: 5′ N₁PyGN₂P 3′. G isguanine. N₁ is any sequence 1 to 6 nucleotides long. In some embodimentsN₁ is at least 50% pyrimidines and preferably at least 50% T. In otherembodiments N₁ includes at least one CG motif, at least one TCG motif,at least one CI motif, at least one TCI motif, at least one IG motif, orat least one TIG motif. N₁ is TCGG or TCGH in other embodiments. H is anucleotide other than G.

Py is a pyrimidine. In some embodiments Py is an unmethylated C.

N₂ is any sequence 0 to 30 nucleotides long. In some embodiments N₂ isat least 50% pyrimidines or is at least 50% T. In other embodiments N₂does not includes any poly G or poly A motifs.

P is a GC-rich palindrome containing sequence at least 10 nucleotideslong. In some embodiments P is completely palindromic. In otherembodiments P is a palindrome having between 1 and 3 consecutiveintervening nucleotides. Optionally the intervening nucleotides may beTG. In other embodiments P includes at least 3, 4, or 5 C and at least3, 4, or 5 G nucleotides. According to other embodiments P includes atleast one Inosine.

In one embodiment the GC-rich palindrome has a base content of at leasttwo-thirds G and C. In another embodiment the GC-rich palindrome has abase content of at least 81 percent G and C. In some embodiments theGC-rich palindrome is at least 12 nucleotides long. The GC-richpalindrome may be made up exclusively of C and G. In some embodimentsthe GC-rich palindrome can include at least one nucleotide that isneither C nor G.

In some embodiments the GC-rich palindrome includes at least one CGGtrimer, at least one CCG trimer, or at least one CGCG tetramer. In someembodiments the GC-rich palindrome includes at least four CGdinucleotides. In certain preferred embodiments the GC-rich palindromehas a central CG dinucleotide.

In certain embodiments the GC-rich palindrome is CGGCGCGCGCCG (SEQ IDNO: 58), CGGCGGCCGCCG (SEQ ID NO: 59), CGACGATCGTCG (SEQ ID NO: 60) orCGACGTACGTCG (SEQ ID NO: 61).

In certain embodiments the GC-rich palindrome is CGCGCGCGCGCG (SEQ IDNO:62), GCGCGCGCGCGC (SEQ ID NO: 63), CCCCCCGGGGGG (SEQ ID NO: 64),GGGGGGCCCCCC (SEQ ID NO: 65), CCCCCGGGGG (SEQ ID NO: 66) or GGGGGCCCCC(SEQ ID NO: 67).

In some embodiments N₁PyGN₂ is a sequence selected from the groupconsisting of TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT,and TCGTCGT.

An immunostimulatory nucleic acid of 13-100 nucleotides in length isprovided according to other aspects of the invention. The nucleic acidhas the formula: 5′ N₁PyG/IN₂P 3′. G/I refers to single nucleotide whichis either a G or an I. G is guanine and I is Inosine.

N₁ is any sequence 1 to 6 nucleotides long. Py is a pyrimidine. N₂ isany sequence 0 to 30 nucleotides long.

P is a palindrome containing sequence at least 10 nucleotides long. Insome embodiments P is a GC-rich palindrome. In other embodiments P is anIC-rich palindrome.

N₁PyIN₂ in some embodiments is TCITCITTTT (SEQ ID NO: 62).

A class of oligonucleotides referred to herein as modified C-classoligonucleotides are characteristically monomeric in solution. It isbelieved that these nucleic acid molecules can form intramolecularduplex structures in vitro, rendering them stable against nucleasedigestion. It is also believed that these same nucleic acid moleculescan form intermolecular duplex and possibly even higher order structureswithin the environment of the intraendosomal compartment, where they arebelieved to exert their biological activity.

Modified C-class oligonucleotides have 3 general formulas. Formula IZ₁ [(X₁Y₁R₁)N(X₂Y₂R₂)_(k)Z₂]_(p)(S₁)_(q)N′(N_(n)) . . . (N₂)(N₁) S₂(N_(1#))(N_(2#)) . . . (N_(n#)) Z₃  (Formula I)wherein each of Z₁, Z₂, and Z₃ is independently any sequence 0 to 12nucleotides long which optionally includes a non-nucleotidic linker orabasic dSpacer; each of X₁ and X₂ is independently a thymidine,deoxyuridine, deoxyadenosine or a 5-substituted deoxyruridine; each ofY₁ and Y₂ is independently a cytosine (C) or a modified cytosine; eachof R₁ and R₂ is independently a guanine (G) or a modified guanine; eachof N and N′ is independently any sequence 0 to 12 nucleotides long whichoptionally includes a non-nucleotidic linker or abasic dSpacer; S₁ is anon-nucleotidic linker, an abasic linker (dSpacers), triethylene glycolunits or hexaethylene glycol units, which optionally provides for 2′5′-,5′5′-, 3′3′-, 2′2′-, or 2′3-internucleoside linkages; S₂ is anynon-palindromic sequence 1 to 10 nucleotides long or a non-nucleotidiclinker, an abasic linker (dSpacers), triethylene glycol units orhexaethylene glycol units; each of N₁, N₂, . . . N_(n), and N_(1#),N_(2#) . . . N_(n#) is any nucleotide or modified nucleotide wherein N₁base-pairs with N_(1#), N₂ base-pairs with N_(2#), . . . and N_(n)base-pairs with N_(n#); k is an integer from 0 to 5; n is an integerfrom 2 to 16; p is an integer from 1 to 6; and q is an integer from 0 to10, and wherein when (N_(n)) . . . (N₂)(N₁) S₂ (N_(1#))(N_(2#)) . . .(N_(n#)) is 10 to 42 nucleotides long, S₂ is 4 to 10 nucleotides long,S₂ comprises a non-nucleotidic linker, an abasic linker (dSpacers),triethylene glycol units or hexaethylene glycol units, and/or (N_(n)) .. . (N₂)(N₁) S₂ (N_(1#))(N_(2#)) . . . (N_(n#)) has a GC content that isless than ⅔.

In one embodiment each of N₁, N₂, . . . N_(n), and N_(1#), N_(2#), . . .N_(n#) is chosen from C, G, or modifications thereof, wherein Cbase-pairs with G.

In one embodiment each of N₁, N₂, . . . N_(n), and N_(1#), N_(2#), . . .N_(1#) is chosen from T, A, or modifications thereof, and T base-pairswith A.

In these and other embodiments each of C, G, A, and T can refer todeoxynucleotides with corresponding bases cytosine, guanine, adenine,and thymine.

In one embodiment each of N₁, N₂ . . . N_(n), and N_(1#), N_(2#), . . .N_(n#) is chosen from C, T, A, G, or modifications thereof, and Cbase-pairs with G, T base-pairs with G, A base-pairs with T, and Abase-pairs with G.

In one embodiment each of N₁, N₂, . . . N_(n), and N_(1#), N_(2#), . . .N_(n#) is chosen from unmodified or modified nucleotides which formWatson-Crick basepairs.

In one embodiment each of N₁, N₂ . . . N_(n), and N_(1#), N_(2#) . . .N_(n#) is chosen from unmodified or modified nucleotides which formnon-Watson-Crick basepairs.

In one embodiment the immunostimulatory nucleic acid molecule includes apartially stabilized backbone with at least one phosphodiester bond.

In one embodiment the immunostimulatory nucleic acid molecule includes abackbone with at least one stabilized internucleotide linkage.

In one embodiment internucleotide linkages of the oligonucleotide areall phosphorothioate linkages.

In one embodiment the immunostimulatory nucleic acid molecule includes apartially stabilized backbone with a phosphodiester bond joining atleast one of Y₁R₁ or Y₂R₂.

In one embodiment Y₁ is C.

In one embodiment R₁ is G.

In one embodiment Y₁ is C and R₁ is G.

In one embodiment X₁ or X₂ is T.

In one embodiment X₁ is T, X₂ is T, Y₁ is C, R₁ is G, and k is 1.

In one embodiment X₁ is T, X₂ is T, Y₁ is C, R₁ is G, k is 1, p is 1, Nand N′ and Z₃ each contain zero nucleotides, and Z₂ is TTTT or d(UUUU).

In one embodiment S₂ is a non-nucleotidic linker.

In one embodiment S₂ contains at least one abasic dSpacer residue.

In one embodiment the oligonucleotide includes at least one branchednon-nucleoside linkage.

In one embodiment the immunostimulatory nucleic acid molecule includesat least one doubler unit, at least one trebler unit, or at least onedoubler unit and at least one trebler unit.

In one embodiment S₁ is a doubler unit or a trebler unit.

In one embodiment the oligonucleotide includes at least one 2′5′-,5′5′-, 3′3′-, 2′2′-, or 2′3′-internucleoside linkage.

In one aspect the invention provides an immunostimulatory nucleic acidmolecule of Formula IIZ₁ (N_(n))(N_(n-1)) . . . (N₂)(N₁)S₂(N_(1#))(N_(2#)) . . .(N_(n-1#))(N_(n#))(S₁)_(q)Z₃[(X₁Y₁R₁)N(X₂Y₂R₂)_(k)Z₂]_(p)  (Formula II)wherein each of Z₁, Z₂, and Z₃ is independently any sequence 0 to 12nucleotides long which optionally includes a non-nucleotidic linker orabasic dSpacer; each of X₁ and X₂ is independently a thymidine,deoxyuridine, deoxyadenosine or a 5-substituted deoxyuridine; each of Y₁and Y₂ is independently a cytosine (C) or a modified cytosine; each ofR₁ and R₂ is independently a guanine (G) or a modified guanine; N is anysequence 0 to 12 nucleotides long which optionally includes anon-nucleotidic linker or abasic dSpacer; S₁ is a non-nucleotidiclinker, an abasic linker (dSpacers), triethylene glycol units orhexaethylene glycol units, which optionally provides for 2′5′-, 5′5′-,3′3′-, 2′2′-, or 2′3′-internucleoside linkages; S₂ is anynon-palindromic sequence 1 to 10 nucleotides long or a non-nucleotidiclinker, an abasic linker (dSpacers), triethylene glycol units orhexaethylene glycol units; each of N₁, N₂, . . . N_(n-1), N_(n), andN_(1#), N_(2#), N_(n-1#), N_(n#) is any nucleotide or modifiednucleotide wherein N₁ base-pairs with N_(1#), N₂ base-pairs with N_(2#),. . . N_(n-1) base-pairs with N_(n-1#), and N_(n) base-pairs withN_(n#); k is an integer from 0 to 5; n is an integer from 2 to 16; p isan integer from 1 to 6; and q is an integer from 0 to 10, and whereinwhen (N_(n)) . . . (N₂)(N₁) S₂ (N_(1#))(N_(2#)) . . . (N_(n#)) is 10 to42 nucleotides long, S₂ is 4 to 10 nucleotides long, S₂ comprises anon-nucleotidic linker, an abasic linker (dSpacers), triethylene glycolunits or hexaethylene glycol units, and/or (N_(n)) . . . (N₂)(N₁)S₂(N_(1#))(N_(2#)) . . . (N_(n#)) has a GC content that is less than ⅔.

In one embodiment Z₁ (N_(n))(N_(n-1)) is TYR, where Y is a cytosine or amodified cytosine and R is a guanine or a modified guanine.

In one embodiment each of N₁, N₂, . . . N_(n-1), N_(n), and N_(1#),N_(2#), . . . N_(n-1#), N_(n#) is chosen from C, G, or modificationsthereof, wherein C base-pairs with G.

In one embodiment each of N₁, N₂, . . . N_(n-1), N_(n), and N_(1#),N_(2#), . . . N_(n-#), N_(n#) is chosen from T, A, or modificationsthereof, and T base-pairs with A.

In these and other embodiments each of C, G, A, and T can refer todeoxynucleotides with corresponding bases cytosine, guanine, adenine,and thymine.

In one embodiment each of N₁, N₂, . . . N_(n-1), N_(n), and N_(1#),N_(2#), . . . N_(n-1#), N_(n#) is chosen from C, T, A, G, ormodifications thereof, and C base-pairs with G, T base-pairs with G, Abase-pairs with T, and A base-pairs with G.

In one embodiment each of N₁, N₂, . . . N_(n-#), N_(n), and N_(1#),N_(2#), . . . N_(n-#), N_(n#), is chosen from unmodified or modifiednucleotides which form Watson-Crick basepairs.

In one embodiment each of N₁, N₂, . . . N_(n-1), N_(n), and N_(1#),N_(2#), . . . N_(n-#), N_(n#) is chosen from unmodified or modifiednucleotides which form non-Watson-Crick basepairs.

In one embodiment the immunostimulatory nucleic acid molecule includes apartially stabilized backbone with at least one phosphodiester bond.

In one embodiment the immunostimulatory nucleic acid molecule includes abackbone with at least one stabilized internucleotide linkage.

In one embodiment internucleotide linkages of the oligonucleotide areall phosphorothioate linkages.

In one embodiment the immunostimulatory nucleic acid molecule includes apartially stabilized backbone with a phosphodiester bond joining atleast one of Y₁R₁ or Y₂R₂.

In one embodiment Y₁ is C.

In one embodiment R₁ is G.

In one embodiment Y₁ is C and R₁ is G.

In one embodiment X₁ or X₂ is T.

In one embodiment X₁ is T, X₂ is T, Y₁ is C, R₁ is G, and k is 1.

In one embodiment X₁ is T, X₂ is T, Y₁ is C, R₁ is G, k is 1, p is 1, Nand N′ and Z₃ each contain zero nucleotides, and Z₂ is TTTT or d(UUUU).

In one embodiment S₂ is a non-nucleotidic linker.

In one embodiment S₂ contains at least one abasic dSpacer residue.

In one embodiment the oligonucleotide includes at least one branchednon-nucleoside linkage.

In one embodiment the immunostimulatory nucleic acid molecule includesat least one doubler unit, at least one trebler unit, or at least onedoubler unit and at least one trebler unit.

In one embodiment S₁ is a doubler unit or a trebler unit.

In one embodiment the oligonucleotide includes at least one 2′5′-,5′5′-, 3′3′-, 2′2′-, or 2′3′-internucleoside linkage.

In one aspect the invention provides an immunostimulatory nucleic acidmolecule of Formula III(U)_(m)Z₃(S₃)  (Formula III)wherein U is Z₁ [(X₁Y₁R₁) N(X₂Y₂R₂)_(k) Z₂]_(p) (S₁)_(q) N′ (N_(n)) . .. (N₃)(N₂)(N₁) S₂ (N_(1#))(N_(2#))(N_(3#)) . . . (N_(n#)); each of Z₁,Z₂, and Z₃ is independently any sequence 0 to 12 nucleotides long whichoptionally includes a non-nucleotidic linker or abasic dSpacer; each ofX₁ and X₂ is independently a thymidine, deoxyuridine, deoxyadenosine ora 5-substituted deoxyuridine; each of Y₁ and Y₂ is independently acytosine or a modified cytosine; each of R₁ and R₂ is independently aguanine or a modified guanine; each of N and N′ is independently anysequence 0 to 12 nucleotides long which optionally includes anon-nucleotidic linker or abasic dSpacer; S₁ is a non-nucleotidiclinker, an abasic linker (dSpacers), triethylene glycol units orhexaethylene glycol units, which optionally provides for 2′5′-, 5′5′-,3′3′-, 2′2′-, or 2′3′-internucleoside linkages; S₂ is anynon-palindromic sequence 1 to 10 nucleotides long or a non-nucleotidiclinker, an abasic linker (dSpacers), triethylene glycol units orhexaethylene glycol units; S₃ is a direct or indirect 2′5′-, 5′5′-,3′3′-, 2′2′-, or 2′3′-internucleoside linkage, or a non-nucleotidiclinker, said non-nucleotidic linker including abasic linkers (dSpacers),triethylene glycol units, or hexaethylene glycol units facilitating a2′5′-, 5′5′-, 3′3′-, 2′2′-, or 2′3′-linkage of m sequence parts; each ofN₁, N₂, . . . N_(n), and N_(1#), N_(2#), . . . N_(n#) is any nucleotideor modified nucleotide wherein N₁ base-pairs with N_(1#), N₂ base-pairswith N_(2#), N₃ base-pairs with N_(3#), . . . and N_(n) base-pairs withN_(n#); k is an integer from 0 to 5; m is an integer from 2 to 10; n isan integer from 2 to 16; p is an integer from 1 to 6; and q is aninteger from 0 to 10.

In certain embodiments Z₁ [(X₁Y₁R₁) N(X₂Y₂R₂)_(k) Z₂]_(p) (S₁)_(q) is anon-palindromic sequence.

In certain embodiments Z₁ [(X₁Y₁R₁) N (X₂Y₂R₂)_(k) Z₂]_(p) (S₁)_(q) isTCGTCGTTTT (SEQ ID NO:29), TCGTCGTTDD (SEQ ID NO:30), TCGA, TCGAC,TCGACGTC, or TCGACGTCG, wherein D is dSpacer.

In certain embodiments Z₁ [(X₁Y₁R₁) N (X₂Y₂R₂)_(k) Z₂]_(p) (S₁)_(q) is apalindromic sequence.

In certain embodiments Z₁ [(X₁Y₁R₁) N (X₂Y₂R₂)_(k) Z₂]_(p) (S₁)_(q) isTCGACGTCGA (SEQ ID NO:31) or TCGTCGACGA (SEQ ID NO:32).

In certain embodiments Z₁ [(X₁Y₁R₁) N (X₂Y₂R₂)_(k) Z₂]_(p) (S₁)_(q) isTCGCGACGTT (SEQ ID NO:33) or TCGCGTCGTT (SEQ ID NO:34).

In one embodiment (N_(n)) . . . (N₂)(N₁) S₂ (N_(1#))(N_(2#)) . . .(N_(n#)) Z₃ includes a sequence AGCGAAGCT, CAATATTTATTG (SEQ ID NO:35),CCGTTTTTGG (SEQ ID NO:36), CGGCGCCGTGCCG (SEQ ID NO:37), CGGCGCCGTTGCCG(SEQ ID NO:38), CGGCGDDCGCCG (SEQ ID NO:39), CGGCGDDDTGCCG (SEQ IDNO:40), CGGCGGDDCCGCCG (SEQ ID NO:41), CGGCGTCGCCGCCG (SEQ ID NO:42),CGTCGACGGGACGGG (SEQ ID NO:43), CGTCGACGTGACGGG (SEQ ID NO:44),GAGAGTTGGGCTCTC (SEQ ID NO:45), GTCGAGGAGGT (SEQ ID NO:46), TAATADDTATTA(SEQ ID NO:47), TAATATCCATTA (SEQ ID NO:48), or TAATATTTATTA (SEQ IDNO:49), wherein D is dSpacer.

In one embodiment (N_(n)) . . . (N₂)(N₁) S₂ (N_(1#))(N_(2#)) . . .(N_(n#)) includes a sequence GGCGCGCTGCCG (SEQ ID NO:50).

In one embodiment the 5′ end of the nucleic acid begins with animmunostimulatory motif chosen from (TCG)_(n)N and RDCGY₁Y₂N. T isthymine, C is unmethylated cytosine, G is guanine, R is a purine, D isnot C, each of Y₁ and Y₂ independently is a pyrimidine, n is an integerbetween 1 and 4, inclusive, and N is any sequence 0-12 bases long.

The 3′ end of the nucleic acid terminates in an inverted repeat capableof forming a hairpin or stem-loop structure. The term “terminates”refers to a structure at or near the 3′ end. Thus, the end of the nearpalindrome may be positioned at the actual 3′ end of the molecule oralternatively the 3′ end may include 1 or more additional nucleotidesthat are not part of the inverted repeat structure. Preferably the 3′end of the molecule includes 3 or fewer nucleotides that do not formpart of the inverted repeat structure.

In one embodiment an “inverted repeat capable of forming a hairpin orstem-loop structure” as used herein refers to a sequence of nucleotidesthat forms a GC-rich stem or hairpin that is 2 to 10 consecutive basepairs long, and includes at least one unmatched or mismatched base. Inindividual embodiments the GC-rich stem is 2, 3, 4, 5, 6, 7, 8, 9, orconsecutive base pairs long. In some embodiments the GC-rich stemincludes at least 2, 3, or 4 G-C base pairs.

In one embodiment an “inverted repeat capable of forming a hairpin orstem-loop structure” as used herein refers to a sequence of nucleotidesthat forms an AT-rich stem or hairpin that is 2 to 10 consecutive basepairs long, and includes at least one unmatched or mismatched base. Inindividual embodiments the AT-rich stem is 2, 3, 4, 5, 6, 7, 8, 9, or 10consecutive base pairs long. In some embodiments the AT-rich stemincludes at least 2, 3, or 4 A-T base pairs.

In some instances the at least one unmatched or mismatched base bridgesthe ends of the stem or hairpin. This may allow the formation of thesecondary structure by providing a flexible point in the molecule forthe stems to base pair and form a hairpin. Alternatively the unmatchedor mismatched base(s) may be within the stem. Preferably if themismatched base is within the stem, then the stem is at least 3basepairs long. The unmatched or mismatched bases(s) may be anynucleotide. In some embodiments the unmatched or mismatched base is a T.Unmatched nucleotides at the end of double-strands are also known asoverhanging nucleotides or dangling ends which can significantlystabilize duplex formation or hairpin formation. Freier S M et al.(1983) Effects of 3′ dangling end stacking on the stability of GGCC andCCGG double helixes. Biochemistry 22:6198-206.

The nucleic acid also includes a partially stabilized backbone includingat least one phosphodiester 5′-CpG-3′ linkage.

In some instances the double-stranded part of the molecule may alsocontain unnatural (non-standard) basepairs (e.g., diaminopyridine pairedwith xanthosinc). Lutz M J et al. (1998) Recognition of a non-standardbase pair by thermostable DNA polymerases. Bioorg Med Chem Lett8:1149-52.

The formulas define subsets of the class of CpG oligonucleotides whichdemonstrated excellent immune stimulating properties. In the formulas 5′refers to the free 5′ end of the oligonucleotide and 3′ refers to thefree 3′ end of the oligonucleotide.

The oligonucleotides may have one or more accessible 5′ or 3′ ends. Insome embodiments a 3′ end can be linked to another 3′ end. Since theimportance of the 5′ and 3′ motifs has been discovered and describedherein, it is also possible to create modified oligonucleotides havingtwo such 5′ or 3′ ends. This may be achieved, for instance by attachingtwo oligonucleotides through a 3′-3′ linkage to generate anoligonucleotide having one or two accessible 5′ ends. Such a structuremight have a formula such as 5′-RDCGY₁Y₂N-NY₂Y₁GCDR-5′ (wherein Drepresents not C; SEQ ID NO:51) or 5′-(TCG)_(n)N—N(GCT)_(n)-5′ (SEQ IDNO:52). The 3′3′- or 5′5′-linkage may be a phosphodiester,phosphorothioate, or any other modified internucleoside bridge. Methodsfor accomplishing such linkages are known in the art. For instance, suchlinkages have been described in Seliger H et al. (1991) Oligonucleotideanalogs with terminal 3′-3′- and 5′-5′-internucleotidic linkages asantisense inhibitors of viral gene expression, Nucleosides & Nucleotides10:469-77 and Jiang Z et al. (1999) Pseudo-cyclic oligonucleotides: invitro and in vivo properties, Bioorg Med Chem 7:2727-35.

In some embodiments the oligonucleotide has one of the followingstructures:

TCGTCGTTTTA, (SEQ ID NO:53) CGGCGCCGTGCCG, (SEQ ID NO:54) CGGCGTCGTGCCG,(SEQ ID NO:55) TCGTCGTTTTACGGCGCCGTGCCG, (SEQ ID NO:56)TCGTCGTTTTACGGCGTCGTGCCG, (SEQ ID NO:57)

The invention in one aspect involves the finding that a specificsub-class of C-class CpG immunostimulatory oligonucleotides having achimeric backbone is highly effective in mediating immune stimulatoryeffects. These CpG oligonucleotides are useful therapeutically andprophylactically for stimulating the immune system to treat cancer,infectious diseases, allergy, asthma, autoimmune disease, and otherdisorders and to help protect against opportunistic infections followingcancer chemotherapy. The strong yet balanced, cellular and humoralimmune responses that result from CpG stimulation reflect the body's ownnatural defense system against invading pathogens and cancerous cells.

The invention involves, in one aspect, the discovery that a subset ofCpG immunostimulatory oligonucleotides have improved immune stimulatoryproperties and reduced renal inflammatory effect. In some instances,renal inflammation has been observed in subjects that have beenadministered a completely phosphorothioate oligonucleotide. It isbelieved that the chimeric oligonucleotides described herein produceless renal inflammation than fully phosphorothioate oligonucleotides.Additionally these oligonucleotides are highly effective in stimulatingan immune response. Thus, the phosphodiester region of the molecule didnot reduce it's affectivity.

The preferred CpG immunostimulatory oligonucleotides fall within one ofthe following 7 general formulas:

5′ TTC_GX₂C_GN₁X₁ _(—) GX₃C_GTT 3′ (SEQ ID NO.: 24) wherein N₁ is 1-3nucleotides in length with N referring to any nucleotide, X₁ is apyrimidine, X₂ and X₃ are any nucleotide.

5′ TTC_GTC_GTTTX₁ _(—) GTC_GTT 3′ (SEQ ID NO.: 25), wherein X₁ is apyrimidine.

(SEQ ID NO.: 25) 5′ T*T*C_G*T*C*T*T*T*X₁_G*T*C_G*T*T 3′,wherein X₁ is a pyrimidine.

5′ TC_GX₁C_G X₂N₁ X₃C_GN₂CG 3′ (SEQ ID NO.: 26), wherein N₁ is 0-3nucleotides in length, N₂ is 0-9 nucleotides in length with N referringto any nucleotide and X₁, X₂, and X₃ are any nucleotide.

5′ TC_GTC_GTN₁TC_GGCGCN₁GCCG 3′ (SEQ ID NO.: 27), wherein N₁ is 0-3nucleotides in length.

(SEQ ID NO.: 27) 5′ T*C_G*T*C_G*T*N₁*T*C_G*G*C*G*CN₁G*C*C*G 3′,wherein N₁ is 0-3 nucleotides in length.

5′ TC_G X₁C_G X₂C_G X₃TC_GGCGC_G N₃3′ (SEQ ID NO.: 28), wherein N₃ is1-5 nucleotides in length with N referring to any nucleotide and X₁, X₂,and X₃ are any nucleotide.

Optionally, when specified in the formula, 5′ refers to the free 5′ endof the oligonucleotide and 3′ refers to the free 3′ end of theoligonucleotide.

The symbol * used in the formulas refers to the presence of a stabilizedinternucleotide linkage. The symbol _ in these structures refers to thepresence of a phosphodiester internucleotide linkage. Theinternucleotide linkages not marked with an * may be stabilized orunstabilized, as long as the oligonucleotide includes at least 2-3phosphodiester or phosphodiester like internucleotide linkages. In someembodiments it is preferred that the oligonucleotides include 3-6phosphodiester or phosphodiester like linkages. In some cases thelinkages between the CG motifs are phosphodiester and in other casesthey are phosphorothioate or other stabilized linkages.

In some embodiment the oligonucleotide has one of the followingstructures:

(SEQ ID NO: 2) T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO:3) T*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G (SEQ ID NO: 4)TC_GTC_GAC_GATC_GGCGC_GCGCCG (SEQ ID NO: 4)T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO: 5)T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T (SEQ ID NO: 6)T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T (SEQ ID NO: 3) TCGTCGTTCGGCGCGCCG(SEQ ID NO: 2) TCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO: 4)TCGTCGACGATCGGCGCGCGCCG (SEQ ID NO: 5) TTCGTCGTTTTGTCGTT (SEQ ID NO: 6)TTTCGTCGTTTCGTCGTT TCGTCGTC CGTCGTCG GTCGTCGT TCGTCGTT CGTCGTTC GTCGTTCGTCGTTCGG CGTTCGGC GTTCGGCG TTCGGCGC TCGGCGCG CGGCGCGC GGCGCGCG GCGCGCGCCGCGCGCC GCGCGCCG. T*C_G*T*C_G*T*C C_G*T*C_G*T*C_G G*T*C_G*T*C_G*TT*C_G*T*C_G*T*T C_G*T*C_G*T*T*C G*T*C_G*T*T*C_G T*C_G*T*T*C_G*GC_G*T*T*C_G*G*C G*T*T*C_G*G*C*G T*T*C_G*G*C*G*C T*C_G*G*C*G*C_GC_G*G*C*G*C_G*C G*G*C*G*C_G*C*G G*C*G*C_G*C*G*C C*G*C_G*C*G*C*CG*C_G*C*G*C*C*G

The terms “nucleic acid” and “oligonucleotide” also encompass nucleicacids or oligonucleotides with substitutions or modifications, such asin the bases and/or sugars. For example, they include oligonucleotideshaving backbone sugars that are covalently attached to low molecularweight organic groups other than a hydroxyl group at the 2′ position andother than a phosphate group or hydroxy group at the 5′ position. Thusmodified oligonucleotides may include a 2′-O-alkylated ribose group. Inaddition, modified oligonucleotides may include sugars such as arabinoseor 2′-fluoroarabinose instead of ribose. Thus the oligonucleotides maybe heterogeneous in backbone composition thereby containing any possiblecombination of polymer units linked together such as peptide-nucleicacids (which have an amino acid backbone with nucleic acid bases).

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymine, 5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,and other naturally and non-naturally occurring nucleobases, substitutedand unsubstituted aromatic moieties. Other such modifications are wellknown to those of skill in the art.

The immunostimulatory oligonucleotides of the instant invention canencompass various chemical modifications and substitutions, incomparison to natural RNA and DNA, involving a phosphodiesterinternucleotide bridge, a β-D-ribose unit and/or a natural nucleotidebase (adenine, guanine, cytosine, thymine, uracil). Examples of chemicalmodifications are known to the skilled person and are described, forexample, in Uhhlmann E et al. (1990) Chem Rev 90:543; “Protocols forOligonucleotides and Analogs” Synthesis and Properties & Synthesis andAnalytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;Crooke S T et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; andHunziker J et al. (1995) Mod Synth Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleotide bridge and/or at a particular β-D-ribose unit and/or ata particular natural nucleotide base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

For example, the invention relates to an oligonucleotide which maycomprise one or more modifications and wherein each modification isindependently selected from:

-   a) the replacement of a phosphodiester internucleotide bridge    located at the 3′ and/or the 5′ end of a nucleotide by a modified    internucleotide bridge,-   b) the replacement of phosphodiester bridge located at the 3′ and/or    the 5′ end of a nucleotide by a dephospho bridge,-   c) the replacement of a sugar phosphate unit from the sugar    phosphate backbone by another unit,-   d) the replacement of a β-D-ribose unit by a modified sugar unit,    and-   e) the replacement of a natural nucleotide base by a modified    nucleotide base.

More detailed examples for the chemical modification of anoligonucleotide are as follows.

A phosphodiester internucleotide bridge located at the 3′ and/or the 5′end of a nucleotide can be replaced by a modified internucleotidebridge, wherein the modified internucleotide bridge is for exampleselected from phosphorothioate, phosphorodithioate,NR¹R²-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate,phosphate-(C₁-C₂₁)—O-alkyl ester,phosphate-[(C₆-C₁₂)aryl-(C₁-C₂₁)—O-alkyl]ester, (C₁-C₈)alkylphosphonateand/or (C₆-C₁₂)arylphosphonate bridges, (C₇-C₁₂)-α-hydroxymethyl-aryl(e.g., disclosed in WO 95/01363), wherein (C₆-C₁₂)aryl, (C₆-C₂₀)aryl and(C₆-C₁₄)aryl are optionally substituted by halogen, alkyl, alkoxy,nitro, cyano, and where R¹ and R² are, independently of each other,hydrogen, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,preferably hydrogen, (C₁-C₈)-alkyl, preferably (C₁-C₄)-alkyl and/ormethoxyethyl, or R¹ and R² form, together with the nitrogen atomcarrying them, a 5-6-membered heterocyclic ring which can additionallycontain a further heteroatom from the group O, S and N.

The replacement of a phosphodiester bridge located at the 3′ and/or the5′ end of a nucleotide by a dephospho bridge (dephospho bridges aredescribed, for example, in Uhlmann E and Peyman A in “Methods inMolecular Biology”, Vol. 20, “Protocols for Oligonucleotides andAnalogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp.355 ff), wherein a dephospho bridge is for example selected from thedephospho bridges formacetal, 3′-thioformacetal, methylhydroxylamine,oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silylgroups.

A sugar phosphate unit (i.e., a β-D-ribose and phosphodiesterinternucleotide bridge together forming a sugar phosphate unit) from thesugar phosphate backbone (i.e., a sugar phosphate backbone is composedof sugar phosphate units) can be replaced by another unit, wherein theother unit is for example suitable to build up a “morpholino-derivative”oligomer (as described, for example, in Stirchak E P et al. (1989)Nucleic Acids Res 17:6129-41), that is, e.g., the replacement by amorpholino-derivative unit; or to build up a polyamide nucleic acid(“PNA”; as described for example, in Nielsen P E et al. (1994) BioconjugChem 5:3-7), that is, e.g., the replacement by a PNA backbone unit,e.g., by 2-aminoethylglycine.

A β-ribose unit or a β-D-2′-deoxyribose unit can be replaced by amodified sugar unit, wherein the modified sugar unit is for exampleselected from β-D-ribose, α-D-2′-deoxyribose, L-2′-deoxyribose,2′-F-2′-deoxyribose, 2′-F-arabinose, 2′-O—(C₁-C₆)alkyl-ribose,preferably 2′-O—(C₁-C₆)alkyl-ribose is 2′-O-methylribose,2′-O—(C₂-C₆)alkenyl-ribose, 2′-[O—(C₁-C₆)alkyl-O—(C₁-C₆)alkyl]-ribose,2′—NH₂-2′-deoxyribose, β-D-xylo-furanose, a-arabinofuranose,2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, forexample, in Froehler J (1992) Am Chem Soc 114:8320) and/or open-chainsugar analogs (described, for example, in Vandendriessche et al. (1993)Tetrahedron 49:7223) and/or bicyclosugar analogs (described, forexample, in Tarkov M et al. (1993) Helv Chim Acta 76:481).

In some preferred embodiments the sugar is 2′-O-methylribose,particularly for one or both nucleotides linked by a phosphodiester orphosphodiester-like internucleotide linkage.

A modified base is any base which is chemically distinct from thenaturally occurring bases typically found in DNA and RNA such as T, C,G, A, and U, but which share basic chemical structures with thesenaturally occurring bases. The modified nucleotide base may be, forexample, selected from hypoxanthine, uracil, dihydrouracil,pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,S—(C₁-C₆)-alkyluracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 2,4-diamino-purine, 8-azapurine, asubstituted 7-deazapurine, preferably 7-deaza-7-substituted and/or7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine,e.g., N4-ethylcytosine, 5-hydroxydeoxycytidine,5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleotides ofnitropyrrole, C5-propynylpyrimidine, and diaminopurine e.g.,2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, hypoxanthine or other modifications of a naturalnucleotide bases. This list is meant to be exemplary and is not to beinterpreted to be limiting.

The oligonucleotides may have one or more accessible 5′ ends. It ispossible to create modified oligonucleotides having two such 5′ ends.This may be achieved, for instance by attaching two oligonucleotidesthrough a 3′-3′ linkage to generate an oligonucleotide having one or twoaccessible 5′ ends. The 3′3′-linkage may be a phosphodiester,phosphorothioate or any other modified internucleotide bridge. Methodsfor accomplishing such linkages are known in the art. For instance, suchlinkages have been described in Seliger, H.; et al., Oligonucleotideanalogs with terminal 3′-3′- and 5′-5′-internucleotidic linkages asantisense inhibitors of viral gene expression, Nucleotides & Nucleotides(1991), 10(1-3), 469-77 and Jiang, et al., Pseudo-cyclicoligonucleotides: in vitro and in vivo properties, Bioorganic &Medicinal Chemistry (1999), 7(12), 2727-2735.

Additionally, 3′ 3′-inked oligonucleotides where the linkage between the3′-terminal nucleotides is not a phosphodiester, phosphorothioate orother modified bridge, can be prepared using an additional spacer, suchas tri- or tetra-ethylenglycol phosphate moiety (Durand, M. et al,Triple-helix formation by an oligonucleotide containing one (dA)12 andtwo (dT)12 sequences bridged by two hexaethylene glycol chains,Biochemistry (1992), 31(38), 9197-204, U.S. Pat. No. 5,658,738, and U.S.Pat. No. 5,668,265). Alternatively, the non-nucleotidic linker may bederived from ethanediol, propanediol, or from an abasic deoxyribose(dSpacer) unit (Fontanel, Marie Laurence et al., Sterical recognition byT4 polynucleotide kinase of non-nucleosidic moieties 5′-attached tooligonucleotides; Nucleic Acids Research (1994), 22(11), 2022-7) usingstandard phosphoramidite chemistry. The non-nucleotidic linkers can beincorporated once or multiple times, or combined with each otherallowing for any desirable distance between the 3′-ends of the two ODNsto be linked.

It recently has been reported that CpG oligonucleotides appear to exerttheir immunostimulatory effect through interaction with Toll-likereceptor 9 (TLR9). Hemmi H et al. (2000) Nature 408.740-5. TLR9signaling activity thus can be measured in response to CpGoligonucleotide or other immunostimulatory oligonucleotide by measuringNF-κB, NF-κB-related signals, and suitable events and intermediatesupstream or downstream of NF-κB.

For use in the instant invention, the oligonucleotides of the inventioncan be synthesized de novo using any of a number of procedures wellknown in the art. For example, the b-cyanoethyl phosphoramidite method(Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981); andnucleotide H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054,1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986, Garegg etal., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let.29:2619-2622, 1988). These chemistries can be performed by a variety ofautomated nucleic acid synthesizers available in the market. Theseoligonucleotides are referred to as synthetic oligonucleotides. Anisolated oligonucleotide generally refers to an oligonucleotide which isseparated from components which it is normally associated with innature. As an example, an isolated oligonucleotide may be one which isseparated from a cell, from a nucleus, from mitochondria or fromchromatin.

The oligonucleotides are partially resistant to degradation (e.g., arestabilized). A “stabilized oligonucleotide molecule” shall mean anoligonucleotide that is relatively resistant to in vivo degradation(e.g. via an exo- or endo-nuclease). Nucleic acid stabilization can beaccomplished via backbone modifications. Oligonucleotides havingphosphorothioate linkages provide maximal activity and protect theoligonucleotide from degradation by intracellular exo- andendo-nucleases. Other modified oligonucleotides include phosphodiestermodified oligonucleotides, combinations of phosphodiester andphosphorothioate oligonucleotide, methylphosphonate,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described (e.g., Uhlmann, E. and Peyman, A., Chem. Rev.90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).

Other stabilized oligonucleotides include: nonionic DNA analogs, such asalkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Oligonucleotides which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

It has been discovered according to the invention that the subsets ofCpG immunostimulatory oligonucleotides have dramatic immune stimulatoryeffects on human cells such as PBMC cells, suggesting that these CpGimmunostimulatory oligonucleotides are effective therapeutic agents forhuman vaccination, cancer immunotherapy, asthma immunotherapy, generalenhancement of immune function, enhancement of hematopoietic recoveryfollowing radiation or chemotherapy, autoimmune disease and other immunemodulatory applications. It has also been demonstrated that the subsetsof CpG immunostimulatory oligonucleotides are useful in vivo for thetreatment of asthma and allergic rhinitis.

A subject having an allergy is a subject that has or is at risk ofdeveloping an allergic reaction in response to an allergen. An allergyrefers to acquired hypersensitivity to a substance (allergen). Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, hay fever, conjunctivitis, bronchial asthma, urticaria (hives)and food allergies, and other atopic conditions.

Allergies are generally caused by IgE antibody generation againstharmless allergens. The cytokines that are induced by systemic ormucosal administration of CpG immunostimulatory oligonucleotides arepredominantly of a class called Th1 (examples are IL-12, IP-10, IFN-αand IFN-γ) and these induce both humoral and cellular immune responses.The other major type of immune response, which is associated with theproduction of IL-4 and IL-5 cytokines, is termed a Th2 immune response.In general, it appears that allergic diseases are mediated by Th2 typeimmune responses. Based on the ability of the CpG immunostimulatoryoligonucleotides to shift the immune response in a subject from apredominant Th2 (which is associated with production of IgE antibodiesand allergy) to a balanced Th2/Th1 response (which is protective againstallergic reactions), an effective dose for inducing an immune responseof a CpG immunostimulatory oligonucleotide as a stand alone therapywithout allergen or in combination with allergen can be administered toa subject to treat or prevent asthma and allergy.

Thus, the CpG immunostimulatory oligonucleotides have significanttherapeutic utility in the treatment of allergic conditions such asasthma and allergic rhinitis. Th2 cytokines, especially IL-4 and IL-5are elevated in the airways of asthmatic subjects. These cytokinespromote important aspects of the asthmatic inflammatory response,including IgE isotope switching, eosinophil chemotaxis and activationand mast cell growth. Th1 cytokines, especially IFN-γ and IL-12, cansuppress the formation of Th2 clones and production of Th2 cytokines.Asthma refers to a disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively associated with atopic or allergic symptoms.

Asthma may be exacerbated by viral infections. The combination of asthmaand viral infections significantly worsens the symptoms in a subject.The oligonucleotides used herein provide significant benefits for thetreatment of viral induced exacerbation of asthma. Several examples ofsuch therapy are presented below.

Allergic rhinitis is a disorder resulting in inflammation of the nasalmucosa caused by allergens such as pollen or dust. The term includesrhinitis medicamentosa, rhinitis sicca, and atrophic rhinitis. There aretwo general types of allergic rhinitis, seasonal and perennial. Seasonalallergic rhinitis is normally referred to as hay fever and is usuallycaused by mould or pollen. Perennial allergic rhinitis is usually causedby an inherent sensitivity to one or more types of allergen. Thiscondition generally continues throughout the year or for as long as thepatient is exposed to the allergen. Both types of allergic rhinitisinvolve a type 1 (IgE-mediated) hypersensitivity that leads toinflammation. This inflammation is thought to be caused by an excessivedegranulation of mast cells and of blood-borne basophils in response tocertain allergens. This leads to increased IgE levels and theconcomitant release of inflammatory mediators, such as histamine, and ofchemotactic factors, such as cytokines, prostaglandins and leukotrienes,that result in a localized inflammatory reaction.

The immunostimulatory oligonucleotides may be administered as standalone therapy without an additional anti-allergy/asthma medicament ortherapy or in combination with such a therapy or medicament. Typicalanti allergy/asthma medicaments and therapies include the use ofintranasal vasoconstrictors, intranasal and systemic antihistamines,intranasal glucocorticoids, mast cell stabilizers, such as cromolyncompounds, and oral decongestants.

An allergen refers to a substance (antigen) that can induce an allergicor asthmatic response in a susceptible subject. The list of allergens isenormous and can include pollens, insect venoms, animal dander dust,fungal spores and drugs (e.g. penicillin). Examples of natural, animaland plant allergens include but are not limited to proteins specific tothe following genuses: Canine (Canis familiaris); Dermatophagoides (e.g.Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosiaartemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata);Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus(Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris);Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietariaofficinalis or Parietaria judaica); Blattella (e.g. Blattellagermanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressussempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus(e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis andJuniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata);Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poacompressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus);Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g.Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g.Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g.Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g.Bromus inermis).

The oligonucleotides are also useful for redirecting an immune responsefrom a Th2 immune response to a Th1 immune response. This results in theproduction of a relatively balanced Th1/Th2 environment. Redirection ofan immune response from a Th2 to a Th1 immune response can be assessedby measuring the levels of cytokines produced in response to the nucleicacid (e.g., by inducing monocytic cells and other cells to produce Th1cytokines, including IL-12, IFN-γ and GM-CSF). The redirection orrebalance of the immune response from a Th2 to a Th1 response isparticularly useful for the treatment or prevention of asthma. Forinstance, an effective amount for treating asthma can be that amount;useful for redirecting a Th2 type of immune response that is associatedwith asthma to a Th1 type of response or a balanced Th1/Th2 environment.Th2 cytokines, especially IL-4 and IL-5 are elevated in the airways ofasthmatic subjects. The CpG immunostimulatory oligonucleotides of theinvention cause an increase in Th1 cytokines which helps to rebalancethe immune system, preventing or reducing the adverse effects associatedwith a predominately Th2 immune response.

The CpG immunostimulatory oligonucleotides are also useful in someaspects of the invention as a vaccine for the treatment of a subject atrisk of developing an infection with an infectious organism or a cancerin which a specific cancer antigen has been identified, in addition toallergy or asthma. The CpG immunostimulatory oligonucleotides can alsobe given without the antigen or allergen for protection againstinfection, allergy or cancer, and in this case repeated doses may allowlonger term protection. A subject at risk as used herein is a subjectwho has any risk of exposure to an infection causing pathogen or acancer or an allergen or a risk of developing cancer. For instance, asubject at risk may be a subject who is planning to travel to an areawhere a particular type of infectious agent is found or it may be asubject who through lifestyle or medical procedures is exposed to bodilyfluids which may contain infectious organisms or directly to theorganism or even any subject living in an area where an infectiousorganism or an allergen has been identified. Subjects at risk ofdeveloping infection also include general populations to which a medicalagency recommends vaccination with a particular infectious organismantigen. If the antigen is an allergen and the subject develops allergicresponses to that particular antigen and the subject may be exposed tothe antigen, i.e., during pollen season, then that subject is at risk ofexposure to the antigen. A subject at risk of developing an allergy toasthma includes those subjects that have been identified as having anallergy or asthma but that don't have the active disease during the CpGimmunostimulatory oligonucleotide treatment as well as subjects that areconsidered to be at risk of developing these diseases because of geneticor environmental factors.

A subject at risk of developing a cancer is one who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission. When asubject at risk of developing a cancer is treated with an antigenspecific for the type of cancer to which the subject is at risk ofdeveloping and a CpG immunostimulatory oligonucleotide, the subject maybe able to kill the cancer cells as they develop. If a tumor begins toform in the subject, the subject will develop a specific immune responseagainst the tumor antigen.

In addition to the use of the CpG immunostimulatory oligonucleotides forprophylactic treatment, the invention also encompasses the use of theCpG immunostimulatory oligonucleotides for the treatment of a subjecthaving an infection, an allergy, asthma, or a cancer.

A subject having an infection is a subject that has been exposed to aninfectious pathogen and has acute or chronic detectable levels of thepathogen in the body. The CpG immunostimulatory oligonucleotides can beused with or without an antigen to mount an antigen specific systemic ormucosal immune response that is capable of reducing the level of oreradicating the infectious pathogen. An infectious disease, as usedherein, is a disease arising from the presence of a foreignmicroorganism in the body. It is particularly important to developeffective vaccine strategies and treatments to protect the body'smucosal surfaces, which are the primary site of pathogenic entry.

A subject having a cancer is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas. In one embodiment the cancer is hairy cellleukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,multiple myeloma, follicular lymphoma, malignant melanoma, squamous cellcarcinoma, renal cell carcinoma, prostate carcinoma, bladder cellcarcinoma, or colon carcinoma.

A subject shall mean a human or vertebrate animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken,primate, e.g., monkey, and fish (aquaculture species), e.g. salmon.Thus, the invention can also be used to treat cancer and tumors,infections, and allergy/asthma in non human subjects. Cancer is one ofthe leading causes of death in companion animals (i.e., cats and dogs).

As used herein, the term treat, treated, or treating when used withrespect to an disorder such as an infectious disease, cancer, allergy,or asthma refers to a prophylactic treatment which increases theresistance of a subject to development of the disease (e.g., toinfection with a pathogen) or, in other words, decreases the likelihoodthat the subject will develop the disease (e.g., become infected withthe pathogen) as well as a treatment after the subject has developed thedisease in order to fight the disease (e.g., reduce or eliminate theinfection) or prevent the disease from becoming worse.

In the instances when the CpG oligonucleotide is administered with anantigen, the subject may be exposed to the antigen. As used herein, theterm exposed to refers to either the active step of contacting thesubject with an antigen or the passive exposure of the subject to theantigen in vivo. Methods for the active exposure of a subject to anantigen are well-known in the art. In general, an antigen isadministered directly to the subject by any means such as intravenous,intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, orsubcutaneous administration. The antigen can be administeredsystemically or locally. Methods for administering the antigen and theCpG immunostimulatory oligonucleotide are described in more detailbelow. A subject is passively exposed to an antigen if an antigenbecomes available for exposure to the immune cells in the body. Asubject may be passively exposed to an antigen, for instance, by entryof a foreign pathogen into the body or by the development of a tumorcell expressing a foreign antigen on its surface.

The methods in which a subject is passively exposed to an antigen can beparticularly dependent on timing of administration of the CpGimmunostimulatory oligonucleotide. For instance, in a subject at risk ofdeveloping a cancer or an infectious disease or an allergic or asthmaticresponse, the subject may be administered the CpG immunostimulatoryoligonucleotide on a regular basis when that risk is greatest, i.e.,during allergy season or after exposure to a cancer causing agent.Additionally the CpG immunostimulatory oligonucleotide may beadministered to travelers before they travel to foreign lands where theyare at risk of exposure to infectious agents. Likewise the CpGimmunostimulatory oligonucleotide may be administered to soldiers orcivilians at risk of exposure to biowarfare to induce a systemic ormucosal immune response to the antigen when and if the subject isexposed to it.

An antigen as used herein is a molecule capable of provoking an immuneresponse. Antigens include but are not limited to cells, cell extracts,proteins, polypeptides, peptides, polysaccharides, polysaccharideconjugates, peptide and non-peptide mimics of polysaccharides and othermolecules, small molecules, lipids, glycolipids, carbohydrates, virusesand viral extracts and multicellular organisms such as parasites andallergens. The term antigen broadly includes any type of molecule whichis recognized by a host immune system as being foreign. Antigens includebut are not limited to cancer antigens, microbial antigens, andallergens.

A cancer antigen as used herein is a compound, such as a peptide orprotein, associated with a tumor or cancer cell surface and which iscapable of provoking an immune response when expressed on the surface ofan antigen presenting cell in the context of an MHC molecule. Cancerantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen, et al.,1994, Cancer Research, 54:1055, by partially purifying the antigens, byrecombinant technology, or by de novo synthesis of known antigens.Cancer antigens include but are not limited to antigens that arerecombinantly expressed, an immunogenic portion of, or a whole tumor orcancer. Such antigens can be isolated or prepared recombinantly or byany other means known in the art.

A microbial antigen as used herein is an antigen of a microorganism andincludes but is not limited to virus, bacteria, parasites, and fungi.Such antigens include the intact microorganism as well as naturalisolates and fragments or derivatives thereof and also syntheticcompounds which are identical to or similar to natural microorganismantigens and induce an immune response specific for that microorganism.A compound is similar to a natural microorganism antigen if it inducesan immune response (humoral and/or cellular) to a natural microorganismantigen. Such antigens are used routinely in the art and are well knownto those of ordinary skill in the art.

The term substantially purified as used herein refers to a polypeptidewhich is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify viral or bacterial polypeptides using standardtechniques for protein purification. The substantially pure polypeptidewill often yield a single major band on a non-reducing polyacrylamidegel. In the case of partially glycosylated polypeptides or those thathave several start codons, there may be several bands on a non-reducingpolyacrylamide gel, but these will form a distinctive pattern for thatpolypeptide. The purity of the viral or bacterial polypeptide can alsobe determined by amino-terminal amino acid sequence analysis. Othertypes of antigens not encoded by a nucleic acid vector such aspolysaccharides, small molecule, mimics etc are included within theinvention.

The oligonucleotides of the invention may be administered to a subjectwith an anti-microbial agent. An anti-microbial agent, as used herein,refers to a naturally-occurring or synthetic compound which is capableof killing or inhibiting infectious microorganisms. The type ofanti-microbial agent useful according to the invention will depend uponthe type of microorganism with which the subject is infected or at riskof becoming infected. Anti-microbial agents include but are not limitedto anti-bacterial agents, anti-viral agents, anti-fungal agents andanti-parasitic agents. Phrases such as “anti-infective agent”,“anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”,“anti-parasitic agent” and “parasiticide” have well-established meaningsto those of ordinary skill in the art and are defined in standardmedical texts. Briefly, anti-bacterial agents kill or inhibit bacteria,and include antibiotics as well as other synthetic or natural compoundshaving similar functions. Antibiotics are low molecular weight moleculeswhich are produced as secondary metabolites by cells, such asmicroorganisms. In general, antibiotics interfere with one or morebacterial functions or structures which are specific for themicroorganism and which are not present in host cells. Anti-viral agentscan be isolated from natural sources or synthesized and are useful forkilling or inhibiting viruses. Anti-fungal agents are used to treatsuperficial fungal infections as well as opportunistic and primarysystemic fungal infections. Anti-parasite agents kill or inhibitparasites.

Examples of anti-parasitic agents, also referred to as parasiticidesuseful for human administration include but are not limited toalbendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl,chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine,diloxanide furoate, eflornithine, furazolidaone, glucocorticoids,halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumineantimoniate, melarsoprol, metrifonate, metronidazole, niclosamide,nifurtimox, oxamniquine, paromomycin, pentamidine isethionate,piperazine, praziquantel, primaquine phosphate, proguanil, pyrantelpamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine,quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin,stibogluconate sodium (sodium antimony gluconate), suramin,tetracycline, doxycycline, thiabendazole, tinidazole,trimethroprim-sulfamethoxazole, and tryparsamide some of which are usedalone or in combination with others.

Antibacterial agents kill or inhibit the growth or function of bacteria.A large class of antibacterial agents is antibiotics. Antibiotics, whichare effective for killing or inhibiting a wide range of bacteria, arereferred to as broad spectrum antibiotics. Other types of antibioticsare predominantly effective against the bacteria of the classgram-positive or gram-negative. These types of antibiotics are referredto as narrow spectrum antibiotics. Other antibiotics which are effectiveagainst a single organism or disease and not against other types ofbacteria, are referred to as limited spectrum antibiotics. Antibacterialagents are sometimes classified based on their primary mode of action.In general, antibacterial agents are cell wall synthesis inhibitors,cell membrane inhibitors, protein synthesis inhibitors, nucleic acidsynthesis or functional inhibitors, and competitive inhibitors.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleotide analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, zidovudine (azidothymidine), imiquimod, andresimiquimod.

The interferons are cytokines which are secreted by virus-infected cellsas well as immune cells. The interferons function by binding to specificreceptors on cells adjacent to the infected cells, causing the change inthe cell which protects it from infection by the virus. α andβ-interferon also induce the expression of Class I and Class II MHCmolecules on the surface of infected cells, resulting in increasedantigen presentation for host immune cell recognition. α andβ-interferons are available as recombinant forms and have been used forthe treatment of chronic hepatitis B and C infection. At the dosageswhich are effective for anti-viral therapy, interferons have severe sideeffects such as fever, malaise and weight loss.

Anti-viral agents useful in the invention include but are not limited toimmunoglobulins, amantadine, interferons, nucleotide analogues, andprotease inhibitors. Specific examples of anti-virals include but arenot limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir;Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin;Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline;Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine;Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; FamotineHydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium;Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal;Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine;Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; SaquinavirMesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime;Zalcitabine; Zidovudine; and Zinviroxime.

Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some anti-fungal agents function as cell wallinhibitors by inhibiting glucose synthase. These include, but are notlimited to, basiungin/ECB. Other anti-fungal agents function bydestabilizing membrane integrity. These include, but are not limited to,immidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g. chitinase) or immunosuppression (501 cream).

CpG immunostimulatory oligonucleotides can be combined with othertherapeutic agents such as adjuvants to enhance immune responses. TheCpG immunostimulatory oligonucleotide and other therapeutic agent may beadministered simultaneously or sequentially. When the other therapeuticagents are administered simultaneously they can be administered in thesame or separate formulations, but are administered at the same time.The other therapeutic agents are administered sequentially with oneanother and with CpG immunostimulatory oligonucleotide, when theadministration of the other therapeutic agents and the CpGimmunostimulatory oligonucleotide is temporally separated. Theseparation in time between the administration of these compounds may bea matter of minutes or it may be longer. Other therapeutic agentsinclude but are not limited to adjuvants, cytokines, antibodies,antigens, etc.

The compositions of the invention may also be administered withnon-nucleic acid adjuvants. A non-nucleic acid adjuvant is any moleculeor compound except for the CpG immunostimulatory oligonucleotidesdescribed herein which can stimulate the humoral and/or cellular immuneresponse. Non-nucleic acid adjuvants include, for instance, adjuvantsthat create a depo effect, immune stimulating adjuvants, and adjuvantsthat create a depo effect and stimulate the immune system.

The CpG immunostimulatory oligonucleotides are also useful as mucosaladjuvants. It has previously been discovered that both systemic andmucosal immunity are induced by mucosal delivery of CpGoligonucleotides. Thus, the oligonucleotides may be administered incombination with other mucosal adjuvants.

Immune responses can also be induced or augmented by theco-administration or co-linear expression of cytokines (Bueler &Mulligan, 1996; Chow et al, 1997; Geissler et al., 1997; Iwasaki et al.,1997; Kim et al., 1997) or B-7 co-stimulatory molecules (Iwasaki et al.,1997; Tsuji et al., 1997) with the CpG immunostimulatoryoligonucleotides. The term cytokine is used as a generic name for adiverse group of soluble proteins and peptides which act as humoralregulators at nano- to picomolar concentrations and which, either undernormal or pathological conditions, modulate the functional activities ofindividual cells and tissues. These proteins also mediate interactionsbetween cells directly and regulate processes taking place in theextracellular environment. Examples of cytokines include, but are notlimited to IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15,IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), interferon-γ (γ-IFN),IFN-α, tumor necrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40ligand.

The oligonucleotides are also useful for improving survival,differentiation, activation and maturation of dendritic cells. The CpGimmunostimulatory oligonucleotides have the unique capability to promotecell survival, differentiation, activation and maturation of dendriticcells.

CpG immunostimulatory oligonucleotides also increase natural killer celllytic activity and antibody dependent cellular cytotoxicity (ADCC). ADCCcan be performed using a CpG immunostimulatory oligonucleotide incombination with an antibody specific for a cellular target, such as acancer cell. When the CpG immunostimulatory oligonucleotide isadministered to a subject in conjunction with the antibody the subject'simmune system is induced to kill the tumor cell. The antibodies usefulin the ADCC procedure include antibodies which interact with a cell inthe body. Many such antibodies specific for cellular targets have beendescribed in the art and many are commercially available.

The CpG immunostimulatory oligonucleotides may also be administered inconjunction with an anti-cancer therapy. Anti-cancer therapies includecancer medicaments, radiation and surgical procedures. As used herein, a“cancer medicament” refers to a agent which is administered to a subjectfor the purpose of treating a cancer, As used herein, “treating cancer”includes preventing the development of a cancer, reducing the symptomsof cancer, and/or inhibiting the growth of an established cancer. Inother aspects, the cancer medicament is administered to a subject atrisk of developing a cancer for the purpose of reducing the risk ofdeveloping the cancer. Various types of medicaments for the treatment ofcancer are described herein. For the purpose of this specification,cancer medicaments are classified as chemotherapeutic agents,immunotherapeutic agents, cancer vaccines, hormone therapy, andbiological response modifiers.

In one embodiment, the cancer medicament is a chemotherapeutic agentselected from the group consisting of methotrexate, vincristine,adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas,5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol,fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan,MM1270, BAY 12-9566, RAS farnesyl transferase inhibitor, farnesyltransferase inhibitor, MMP, MTA/LY231514, LY264618Lometexol, Glamolec,CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833,Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556,CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805,DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Placlitaxel, Taxol/Paclitaxel,Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel,Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358(774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751/oralplatinum, UFT (Tegafur/Uracil), Ergamisol/Levamisole,Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan,Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU79553/Bis-Naphthalimide, LU 103793/Dolastain, Caetyx/liposomaldoxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds,CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide,Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin,Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel,prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylatingagents such as melphelan and cyclophosphamide, Aminoglutethimide,Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl,Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane(o.p′-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastinesulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin,Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′deoxycoformycin),Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate. In animportant embodiment, the cancer medicament is taxol. In anotherembodiment the cancer medicament is a combination of carboplatin andpaclitaxel.

In another embodiment, the cancer medicament is an immunotherapeuticagent selected from the group consisting of Ributaxin, Herceptin,Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN,Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94,anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE,Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide,CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2,MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab and ImmuRAIT-CEA.

Additionally, the methods of the invention are intended to embrace theuse of more than one cancer medicament along with the CpGimmunostimulatory oligonucleotides. As an example, where appropriate,the CpG immunostimulatory oligonucleotides may be administered with botha chemotherapeutic agent and an immunotherapeutic agent. Alternatively,the cancer medicament may embrace an immunotherapeutic agent and acancer vaccine, or a chemotherapeutic agent and a cancer vaccine, or achemotherapeutic agent, an immunotherapeutic agent and a cancer vaccineall administered to one subject for the purpose of treating a subjecthaving a cancer or at risk of developing a cancer.

The use of CpG immunostimulatory oligonucleotides in conjunction withimmunotherapeutic agents such as monoclonal antibodies is able toincrease long-term survival through a number of mechanisms includingsignificant enhancement of ADCC (as discussed above), activation ofnatural killer (NK) cells and an increase in IFNα levels. Theoligonucleotides when used in combination with monoclonal antibodiesserve to reduce the dose of the antibody required to achieve abiological result.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses.

The CpG immunostimulatory oligonucleotides are also useful for treatingand preventing autoimmune disease. Autoimmune disease is a class ofdiseases in which a subject's own antibodies react with host tissue orin which immune effector T cells are autoreactive to endogenous selfpeptides and cause destruction of tissue. Thus an immune response ismounted against a subject's own antigens, referred to as self antigens.Autoimmune diseases include but are not limited to rheumatoid arthritis,Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE),autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto'sthyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigusvulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, scleroderma with anti-collagen antibodies,mixed connective tissue disease, polymyositis, pernicious anemia,idiopathic Addison's disease, autoimmune-associated infertility,glomerulonephritis (e.g., crescentic glomerulonephritis, proliferativeglomerulonephritis), bullous pemphigoid, Sjögren's syndrome, insulinresistance, and autoimmune diabetes mellitus.

A “self-antigen” as used herein refers to an antigen of a normal hosttissue. Normal host tissue does not include cancer cells. Thus an immuneresponse mounted against a self-antigen, in the context of an autoimmunedisease, is an undesirable immune response and contributes todestruction and damage of normal tissue, whereas an immune responsemounted against a cancer antigen is a desirable immune response andcontributes to the destruction of the tumor or cancer. Thus, in someaspects of the invention aimed at treating autoimmune disorders it isnot recommended that the CpG immunostimulatory oligonucleotides beadministered with self antigens, particularly those that are the targetsof the autoimmune disorder.

In other instances, the CpG immunostimulatory oligonucleotides may bedelivered with low doses of self-antigens. A number of animal studieshave demonstrated that mucosal administration of low doses of antigencan result in a state of immune hyporesponsiveness or “tolerance.” Theactive mechanism appears to be a cytokine-mediated immune deviation awayfrom a Th1 towards a predominantly Th2 and Th3 (i.e., TGF-β dominated)response. The active suppression with low dose antigen delivery can alsosuppress an unrelated immune response (bystander suppression) which isof considerable interest in the therapy of autoimmune diseases, forexample, rheumatoid arthritis and SLE. Bystander suppression involvesthe secretion of Th1-counter-regulatory, suppressor cytokines in thelocal environment where proinflammatory and Th1 cytokines are releasedin either an antigen-specific or antigen-nonspecific manner. “Tolerance”as used herein is used to refer to this phenomenon. Indeed, oraltolerance has been effective in the treatment of a number of autoimmunediseases in animals including: experimental autoimmune encephalomyelitis(EAE), experimental autoimmune myasthenia gravis, collagen-inducedarthritis (CIA), and insulin-dependent diabetes mellitus. In thesemodels, the prevention and suppression of autoimmune disease isassociated with a shift in antigen-specific humoral and cellularresponses from a Th1 to Th21Th3 response.

The invention also includes a method for inducing antigen non-specificinnate immune activation and broad spectrum resistance to infectiouschallenge using the CpG immunostimulatory oligonucleotides. The termantigen non-specific innate immune activation as used herein refers tothe activation of immune cells other than B cells and for instance caninclude the activation of NK cells, T cells or other immune cells thatcan respond in an antigen independent fashion or some combination ofthese cells. A broad spectrum resistance to infectious challenge isinduced because the immune cells are in active form and are primed torespond to any invading compound or microorganism. The cells do not haveto be specifically primed against a particular antigen. This isparticularly useful in biowarfare, and the other circumstances describedabove such as travelers.

The CpG immunostimulatory oligonucleotides may be directly administeredto the subject or may be administered in conjunction with a nucleic aciddelivery complex. A nucleic acid delivery complex shall mean a nucleicacid molecule associated with (e.g. ionically or covalently bound to; orencapsulated within) a targeting means (e.g. a molecule that results inhigher affinity binding to target cell. Examples of nucleic aciddelivery complexes include nucleic acids associated with a sterol (e.g.cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), ora target cell specific binding agent (e.g. a ligand recognized by targetcell specific receptor). Preferred complexes may be sufficiently stablein vivo to prevent significant uncoupling prior to internalization bythe target cell. However, the complex can be cleavable under appropriateconditions within the cell so that the oligonucleotide is released in afunctional form.

Delivery vehicles or delivery devices for delivering antigen andoligonuclotides to surfaces have been described. The CpGimmunostimulatory oligonucleotide and/or the antigen and/or othertherapeutics may be administered alone (e.g., in saline or buffer) orusing any delivery vehicles known in the art.

The term effective amount of a CpG immunostimulatory oligonucleotiderefers to the amount necessary or sufficient to realize a desiredbiologic effect. For example, an effective amount of a CpGimmunostimulatory oligonucleotide administered with an antigen forinducing mucosal immunity is that amount necessary to cause thedevelopment of IgA in response to an antigen upon exposure to theantigen, whereas that amount required for inducing systemic immunity isthat amount necessary to cause the development of IgG in response to anantigen upon exposure to the antigen. Combined with the teachingsprovided herein, by choosing among the various active compounds andweighing factors such as potency, relative bioavailability, patient bodyweight, severity of adverse side-effects and preferred mode ofadministration, an effective prophylactic or therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject. The effectiveamount for any particular application can vary depending on such factorsas the disease or condition being treated, the particular CpGimmunostimulatory oligonucleotide being administered the size of thesubject, or the severity of the disease or condition. One of ordinaryskill in the art can empirically determine the effective amount of aparticular CpG immunostimulatory oligonucleotide and/or antigen and/orother therapeutic agent without necessitating undue experimentation.

Subject doses of the compounds described herein for mucosal or localdelivery typically range from about 0.1 μg to 10 mg per administration,which depending on the application could be given daily, weekly, ormonthly and any other amount of time there between. More typicallymucosal or local doses range from about 10 μg to 5 mg peradministration, and most typically from about 100 μg to 1 mg, with 2-4administrations being spaced days or weeks apart. More typically, immunestimulant doses range from 1 μg to 10 mg per administration, and mosttypically 10 μg to 1 mg, with daily or weekly administrations. Subjectdoses of the compounds described herein for parenteral delivery for thepurpose of inducing an antigen-specific immune response, wherein thecompounds are delivered with an antigen but not another therapeuticagent are typically 5 to 10,000 times higher than the effective mucosaldose for vaccine adjuvant or immune stimulant applications, and moretypically 10 to 1,000 times higher, and most typically 20 to 100 timeshigher. Doses of the compounds described herein for parenteral deliveryfor the purpose of inducing an innate immune response or for increasingADCC or for inducing an antigen specific immune response when the CpGimmunostimulatory oligonucleotides are administered in combination withother therapeutic agents or in specialized delivery vehicles typicallyrange from about 0.1 μg to 10 mg per administration, which depending onthe application could be given daily, weekly, or monthly and any otheramount of time there between. More typically parenteral doses for thesepurposes range from about 10 μg to 5 mg per administration, and mosttypically from about 100 μg to 1 mg, with 2-4 administrations beingspaced days or weeks apart. In some embodiments, however, parenteraldoses for these purposes may be used in a range of 5 to 10,000 timeshigher than the typical doses described above.

For any compound described herein the therapeutically effective amountcan be initially determined from animal models. A therapeuticallyeffective dose can also be determined from human data for CpGoligonucleotides which have been tested in humans (human clinical trialshave been initiated) and for compounds which are known to exhibitsimilar pharmacological activities, such as other adjuvants, e.g., LTand other antigens for vaccination purposes. Higher doses may berequired for parenteral administration. The applied dose can be adjustedbased on the relative bioavailability and potency of the administeredcompound. Adjusting the dose to achieve maximal efficacy based on themethods described above and other methods as are well-known in the artis well within the capabilities of the ordinarily skilled artisan.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

For use in therapy, an effective amount of the CpG immunostimulatoryoligonucleotide can be administered to a subject by any mode thatdelivers the oligonucleotide to the desired surface, e.g., mucosal,systemic. Administering the pharmaceutical composition of the presentinvention may be accomplished by any means known to the skilled artisan.Preferred routes of administration include but are not limited to oral,parenteral, intramuscular, intranasal, sublingual, intratracheal,inhalation, ocular, vaginal, and rectal.

For oral administration, the compounds (i.e., CpG immunostimulatoryoligonucleotides, antigens and other therapeutic agents) can beformulated readily by combining the active compound(s) withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.Pharmaceutical preparations for oral use can be obtained as solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Optionally the oral formulations may also be formulated insaline or buffers, i.e. EDTA for neutralizing internal acid conditionsor may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the abovecomponent or components. The component or components may be chemicallymodified so that oral delivery of the derivative is efficacious.Generally, the chemical modification contemplated is the attachment ofat least one moiety to the component molecule itself, where said moietypermits (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the component or components and increase incirculation time in the body. Examples of such moieties include:polyethylene glycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski and Davis, 1981, “SolublePolymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg and Roberts,eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al.,1982, J. Appl. Biochem. 4:185-189. Other polymers that could be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the component the location of release may be the stomach, the smallintestine (the duodenum, the jejunum, or the ileum), or the largeintestine. One skilled in the art has available formulations which willnot dissolve in the stomach, yet will release the material in theduodenum or elsewhere in the intestine. Preferably, the release willavoid the deleterious effects of the stomach environment, either byprotection of the oligonucleotide or by release of the biologicallyactive material beyond the stomach environment, such as in theintestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theoligonucleotide may be formulated (such as by liposome or microsphereencapsulation) and then further contained within an edible product, suchas a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential non-ionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the oligonucleotideor derivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the oligonucleotides(or derivatives thereof). The oligonucleotide is delivered to the lungsof a mammal while inhaling and traverses across the lung epitheliallining to the blood stream. Other reports of inhaled molecules includeAdjei et al., 1990, Pharmaceutical Research, 7:565-569; Adjei et al.,1990, International Journal of Pharmaceutics, 63:135-144 (leuprolideacetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology,13(suppl. 5):143-146 (endothelin-1); Hubbard et al., 1989, Annals ofInternal Medicine, Vol. III, pp. 206-212 (al-antitrypsin); Smith et al.,1989, J. Clin. Invest. 84:1145-1146 (a-1-proteinase); Oswein et al.,1990, “Aerosolization of Proteins”, Proceedings of Symposium onRespiratory Drug Delivery II, Keystone, Colo., March, (recombinant humangrowth hormone); Debs et al., 1988, J. Immunol. 140:3482-3488(interferon-g and tumor necrosis factor alpha) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor). A method andcomposition for pulmonary delivery of drugs for systemic effect isdescribed in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong etal.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of oligonucleotide (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvants and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified oligonucleotidemay also be prepared in different formulations depending on the type ofchemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise oligonucleotide dissolved in waterat a concentration of about 0.1 to 25 mg of biologically activeoligonucleotide per mL of solution. The formulation may also include abuffer and a simple sugar (e.g., for oligonucleotide stabilization andregulation of osmotic pressure). The nebulizer formulation may alsocontain a surfactant, to reduce or prevent surface induced aggregationof the oligonucleotide caused by atomization of the solution in formingthe aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the oligonucleotidesuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing oligonucleotide and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, or mannitolin amounts which facilitate dispersal of the powder from the device,e.g., 50 to 90% by weight of the formulation. The oligonucleotide shouldmost advantageously be prepared in particulate form with an averageparticle size of less than 10 mm (or microns), most preferably 0.5 to 5mm, for most effective delivery to the distal lung.

Nasal delivery of a pharmaceutical composition of the present inventionis also contemplated. Nasal delivery allows the passage of apharmaceutical composition of the present invention to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed, The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed. The opening is usually found in the top of the bottle, and thetop is generally tapered to partially fit in the nasal passages forefficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The CpG immunostimulatory oligonucleotides and optionally othertherapeutics and/or antigens may be administered per se (neat) or in theform of a pharmaceutically acceptable salt. When used in medicine thesalts should be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: hydrochloric, hydrobromic,sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulphonic, tartaric, citric, methane sulphonic, formic, malonic,succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the invention contain an effectiveamount of a CpG immunostimulatory oligonucleotide and optionallyantigens and/or other therapeutic agents optionally included in apharmaceutically-acceptable carrier. The termpharmaceutically-acceptable carrier means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm carrier denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Oligodeoxynucleotides (ODNs)

The following ODN are used in the examples.

SEQ ID- NO. Sequence  1 T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G  2T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G  3T*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G  4T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G  5T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T  6 T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T 7 T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T  8 TCG TCG TTT TGTCGT TTT GTC GTT (all bonds *)  9T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 10 TCG TCG TTT TGA CGT TTTGTC GTT (all bonds *) 14 TCCAGGACTTCTCTCAGGTT (all bonds *) 15C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 16G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 17T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 18C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 19G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 20 T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G21 T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C 22T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C 23T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*CMaterials and Methods:Oligodeoxynucleotides (ODNs)

ODNs were purchased from Biospring (Frankfurt, Germany) or Sigma-Ark(Darmstadt, Germany), and were controlled for identity and purity byColey Pharmaceutical GmbH (Langenfeld, Germany). ODNs were diluted inphosphate-buffered saline (Sigma, Germany), and stored at −20° C. Alldilutions were carried out using pyrogen-free reagents. SEQ ID NO. 15-23ODNs were synthesized by Trilink Biotechnologies.

Cell Purification

Peripheral blood buffy coat preparations from healthy male and femalehuman donors were obtained from the Blood Bank of the University ofDüsseldorf (Germany) and from these, PBMC were purified bycentrifugation over Ficoll-Hypaque (Sigma). The purified PBMC wereeither used freshly (for most assays) or were suspended in freezingmedium and stored at −70° C. When required, aliquots of these cells werethawed, washed and resuspended in RPMI 1640 culture medium(BioWhittaker, Belgium) supplemented with 5% (v/v) heat inactivatedhuman AB serum (BioWhittaker, Belgium) or 10% (v/v) heat inactivatedFCS, 2 mM L-glutamine (BioWhittaker), 100 U/ml penicillin and 100 μg/mlstreptomycin (Invitrogen (Karlsruhe, Germany)).

Cytokine Detection

Thawed or fresh PBMC were seeded on 48 well flat-bottom plates, or 96well round-bottom plates, and incubated with ODN in the concentrationsas indicated in a humidified incubator at 37° C. Culture supernatantswere collected and if not used immediately, were frozen at −20° C. untilrequired. Amounts of cytokines in the supernatants were assessed usingcommercially available ELISA Kits (Diaclone, USA) or in-house ELISAsdeveloped using commercially available antibodies (from BectonDickinson/Pharmingen or PBL).

Studies for Example 13 were conducted as follows:

Spleens were removed from 6 mice (male, BALB/c). The splenocytes fromeach spleen were separated by pushing gently through a cell sieve (70 μmpore size), and were then pooled. Splenocytes were added to wells ofculture plates. 1×10⁷ cells in a volume of 900 μl medium were added toeach well. Medium was RPMI 1640 containing 10% fetal bovine serum. 100μl aliquots of CpG ODN solutions in medium were added to each well togive final concentrations in the wells of 0.01-10 μg/ml. Afterincubation for 36 hours (37 C, 5% CO₂ incubator), culture supernatantswere collected and assayed for cytokine concentrations using either theLuminex multiplex assay (IFNγ, IP-10, IL-10, IL-6, TNFα) or ELISA(IFNα).

Induction of Antigen-Induced Increase in Nasal Resistance in Guinea Pigs

Guinea pigs (male, Hartley) were sensitized with antigen (ovalbumin, 5mg both intraperitoneal and subcutaneous) on study day 0. A boostsensitization (5 mg, intraperitoneal) was given on study day 4. Guineapigs were antigen challenged by exposure to intranasally-administeredantigen twice each week for two consecutive weeks. The first challengewas on study day 14. SEQ ID NO:7 (lot number AQE-03J-001-M, 0.03-1 mg/kgin 15011/kg saline) was administered intranasally once each week, twodays before the first antigen challenge of the week. With the exceptionof the final challenge on study day 24, antigen challenge was withovalbumin (1.5 mg/kg in 150 μl/kg saline). Animals were pretreated withmepyramine (10 mg/kg, intraperitoneal) 30 minutes before challenge toprotect against histamine-induced anaphylaxis. On study day 24, guineapigs were anesthetized to allow measurement of nasal resistance using aBuxco respiratory mechanics system and software. The final antigenchallenge was then made with ovalbumin (2.5 mg in 250 μl saline)delivered into the nasopharynx. Animals were pretreated with mepyramine(3 mg/kg, intraperitoneal) 30 minutes before challenge. Nasal resistancewas measured for 40 minutes after challenge.

Induction of Antigen-Induced Increase in Nasal Resistance in Mice

Mice (male BALB/c) were sensitized on study days 0 and 7 with antigen(ovalbumin, 100 μg, i.p.) with aluminum hydroxide adjuvant (Pierce Alumi.p.). Mice were antigen challenged daily by exposure tointranasally-administered antigen (1 mg in 10 μl saline). The firstchallenge was on study day 14. SEQ ID NO:7 was administered intranasallytwice. The first dose was given 2 days before the first antigenchallenge. The second dose was given 7 days later. Alternatively,budesonide (an anti-inflammatory, synthetic corticosteroid) wasadministered intranasally daily. The first dose was given 2 days beforethe first antigen challenge. On each day, the budesonide dose wasadministered intranasally 4 hours before intranasal antigen challenge.Endpoints were measured on study day 26 (i.e. 7 days after the seconddose of SEQ ID NO: 7. Nasal tissues were taken for histopathologicalassessment of inflammation. Separate mice received a final antigenchallenge and incidences of sneezing and nasal rubbing were counted fora 10 minute period after challenge.

Statistical Analysis

The Mann-Whitney test was used for comparison of data sets where thepopulations being compared were not normal. The Dunnitt multiplecomparisons test was used for comparison of data sets to a singlecontrol.

Cytokine Induction in the Mouse In Vivo

Mice (male, BALB/c) were dosed with CpG ODNs (0.1 and 1 mg/kg) orcontrol vehicle (saline, 25 μl) by intranasal instillation.Bronchoalveolar lavage fluid and serum (separated from blood obtained bycardiac puncture) were collected 8 hours and 15 hours after dosing. Cellnumbers in bronchoalveolar lavage fluid were counted with an Adviaautomated cell counter. Concentrations of cytokines and chemokines inbronchoalveolar lavage fluid and serum were assayed using either ELISA(IFNα, IL-12p40) or the Luminex cytokine multiplex system (IFNγ, IP-10,IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13, IL-15,IL-17, GM-CSF, RANTES, TNFα). Minimum detectable concentrations weredifferent for each analyte, but were in the range 0.3-12 pg/ml.

Influenza Virus Induction of Inflammation in Mouse Airways.

Influenza virus (influenza type A, subtype H1N1, mouse adapted strainPR8) was a gift from David Woodhouse, Trudeau Institute, Saranac Lake,N.Y. As a preliminary study to titrate influenza dose and determinetime-course of infection, BALB/c mice (female) were infected withinfluenza virus by intranasal instillation on study day 0. Mice received50, 200 or 500 egg-infective doses (ED)₅₀ of virus in 40 μl saline.Airway inflammation was assessed 1, 3, 6, 9 and 14 days after infection.

Intranasal Administration of CpG ODN and Measurement of AirwayInflammation

Mice received a CpG ODN (0.03, 0.3 or 3 mg/kg) by intranasalinstillation in 25 μl saline. Each mouse was dosed twice, 6 days and 2days before infection with influenza virus (200 EID₅₀, intranasal). Thisvirus dose was selected from the preliminary study. Airway inflammationand virus load in the lung were assessed 6 days after virus infection.This time point was selected from the preliminary study. Cells inairways were recovered by bronchoalveolar lavage. Total leukocyte countswere made with an Advia automated cell counter (Adiva, BayerDiagnostics, Zurich, Switzerland). Differential cell counts were made bylight microscopy of cytocentrifuge preparations stained withWright-Giemza stain. Lungs were removed and homogenized with 300 μlsterile PBS. supernatant was collected and virus load was assayed usingan enzyme immunoassay kit (Takara Biomedical, Shiga, Japan) used tomanufacturer's instructions. This assay utilizes a monoclonal antibodyagainst influenza A virus nuclear protein as solid phase, and apolyclonal anti-influenza virus detection antibody.

Example 1 Effects on IFN-α Secretion by Human PBMC Treated with CpG ODN

Methods: Human peripheral blood mononuclear cells from either three (SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6) or seven (SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3) donors were incubated with CpG ODN for 48 hours atconcentrations indicated. Interferon-alpha secretion by human PBMC wasmeasured.

Results: FIG. 1 demonstrates increased production of IFN-α uponincubation with CpG ODN. Data represent the mean +/−SEM. Note that theabsolute levels in pg/ml can not be compared directly, as PBMC fromdifferent donors were used and results from each donor are variable.

Example 2 Stimulation of TLR9-Transfected Cells In Vitro

Methods: HEK 293 cells transfected with human TLR9 were incubated withSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQID NO:6 for 16 hours. The signal was determined by a luciferase readout.

Results: The results are shown in Table 1. The EC50 was calculated usingSigma Plot (SigmaPlot 2002 for Windows version 8.0). The maximalstimulation index (max SI) was calculated as the quotient between thehighest value of all concentrations tested for any ODN and the mediumcontrol.

TABLE 1 ODN EC50 max SI SEQ ID NO: 1 2320 22 SEQ ID NO: 2 4730 20 SEQ IDNO: 3 2400 16 SEQ ID NO: 4 3200 14 SEQ ID NO: 5 4290 13 SEQ ID NO: 64580 11

Example 3 Effects of the CpG Oligodeoxynucleotide SEQ ID NO:7 AgainstAntigen-Induced Increase in Nasal Resistance in Guinea Pigs

Methods: To investigate the effects of the CpG oligodeoxynucleotide SEQID NO:7 against antigen induced increases in nasal resistance in theguinea pigs guinea pigs were sensitized with antigen, thenantigen-challenged nasally. Nasal resistance was measured for 40 minutesafter challenge.

TABLE 2 Summary of study protocol Dose Dose with with Antigen SEQ IDAntigen SEQ ID Antigen sensitize NO: 7 challenge NO: 7 challenge Day: 04 12 14 17 19 21 24 Measure antigen-induced increase in nasal resistance

Results: FIG. 2 demonstrates that antigen challenge caused a progressiveincrease in nasal resistance over 40 minutes that was significantlysuppressed in guinea pigs that had been treated with SEQ ID NO:7 (0.03-1mg/kg).

Example 4 Effects of CpG Oligonucleotide SEQ ID NO:7 in a Mouse Model ofAllergic Rhinitis

Methods: BALB/c mice were used to study the effects of SEQ ID NO:7 onsymptoms of allergic rhinitis. After sensitization and antigenchallenge, nasal tissues were taken for histopathological assessment ofinflammation. Separate mice received a final antigen challenge andincidences of sneezing and nasal rubbing were counted for a 10 minuteperiod after challenge.

TABLE 3 Summary of study protocol: oligonucleotide-treated mice SEQ IDNO: 7 SEQ ID NO: 7 or SEQ ID NO: 8 Sensitize or SEQ ID NO: 8 Dailyantigen challenges Day: 0 7 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26Endpoints

TABLE 4 Summary of study protocol: budesonide-treated mice Daily doseswith budesonide Sensitize Daily antigen challenges Day: 0 7 12 13 14 1516 17 18 19 20 21 22 23 24 25 26 Endpoints

Results: Antigen challenge caused sneezing and nasal rubbing. Asdemonstrated in FIG. 3 the incidences of both were suppressed in micetreated with SEQ ID NO:7.

Example 5 Influenza Virus-Induced Airway Inflammation in Mouse Lung:Effects of Class C CpG Oligodeoxynucleotides

Introduction: CpG oligodeoxynucleotides (ODNs) induce immune-modifyingcytokines that should provide anti-asthma effects. Class C CpG ODNsinduce higher titers of IFNα than previous class B ODNs. Class C CpGODNs may offer the additional benefit of suppressing virus-inducedexacerbations of asthma. This study investigated the ability of threeclass C CpG ODNs to suppress virus (influenza) load in mouse lungs andvirus-induced airways inflammation.

Methods: Influenza virus was used to induce airway inflammation inBALB/c mice. CpG ODNs were administered intranasally and the protectiveeffect was measured.

Results: FIG. 4 shows that in a preliminary study to titrate influenzadose and determine time-course of infection, influenza virus caused anaccumulation of leukocytes in the airways. Peak inflammation wasachieved after 6-9 days. Mice infected with 500 EID50 of virus showedmarked weight loss after 6 days and were sacrificed.

FIG. 5 demonstrates the protective effects of CpG ODNs on virus load andvirus-induced airways inflammation. Pretreatment with CpG ODNs beforeinfection with influenza virus (200 EID50) reduced virus load in thelung as assayed 6 days after infection. FIG. 6 demonstrates thatinfection with influenza virus caused an accumulation of leukocytes inthe airways 6 days later. These were predominantly neutrophils andmononuclear cells (monocytes, macrophages and lymphocytes). There weevery few eosinophils. Cell accumulation was significantly suppressed inmice pretreated with any of the CpG ODNs.

Example 6 Effects of class C CpG Oligodeoxynucleotides AgainstAntigen-Induced Airways Inflammation in the Mouse

The activity of three class C CpG oligodeoxynucleotides (ODNs) wascompared. The class B CpG ODN SEQ ID NO:7 was included in the study forcomparison.

Methods: Mice (male BALB/c) were sensitized on study days 0 and 7 withantigen (cockroach, 10 μg, intraperitoneal) with aluminum hydroxideadjuvant (Pierce Alum, intraperitoneal). Mice were antigen challenged byexposure to intranasally-administered antigen (10 μg in 40 μl saline),twice each week for two consecutive weeks. The first challenge was onstudy day 21. CpG ODNs (1, 10 and 100 μg/kg) were administeredintranasally once each week, two days before the first antigen challengeof the week.

Airways inflammation was assessed 48 hours after the last antigenchallenge. Cells in airways were recovered by bronchoalveolar lavage.Differential cell counts were made by an Advia automated cell counter.Numbers of T cells (Total CD3+ cells, and CD3+ CD4+ cells) were countedby flow cytometry.

TABLE 5 Summary of study protocol Dose Dose with with Antigen CpGAntigen CpG Antigen sensitize ODN challenge ODN challenge Day: 0 7 19 2124 26 28 31 33 Assess airways inflammation

TABLE 6 CpG ODNs tested SEQ ID NO: 7 Semi-soft class B Lot A25-0313-L1SEQ ID NO: 1 Semi-soft class C Lot C44-1209-M1B SEQ ID NO: 2 Semi-softclass C Lot C44-1209-M2D SEQ ID NO: 3 Semi-soft class C Lot C44-1209-M4B

Results: Antigen challenge caused accumulations of eosinophils and Tcells in the airways (FIGS. 7 and 8). There was no accumulation ofneutrophils. Each of the CpG ODNs caused a significant suppression ofeosinophil accumulation at the highest dose tested (100 mg/kg) (FIG. 7).Numbers of T cells were also lower, although the reductions were notgenerally statistically significant (FIG. 8).

Example 7 Cytokine Induction by Class C CpG Oligodeoxynucleotides in theMouse in Vivo

The activities of three class C CpG oligodeoxynucleotides (ODNs) werecompared. The class B CpG ODN SEQ ID NO:7 was included in the study forcomparison.

Methods: Concentrations of cytokines and chemokines in bronchoalveolarlavage fluid and serum were assayed as described in materials andmethods.

TABLE 7 Treatment groups Time points for Intranasal sample CpGoligodeoxynucleotide doses collection Vehicle 8, 15 hours SEQ ID NO: 7Semi-soft class B Lot A25-0313-L1 0.1, 1 mg/kg 8, 15 hours ODN SEQ IDNO: 1 Semi-soft class C Lot C44-1209-M1B 0.1, 1 mg/kg 8, 15 hours ODNSEQ ID NO: 2 Semi-soft class C Lot C44-1209-M2D 0.1, 1 mg/kg 8, 15 hoursODN SEQ ID NO: 3 Semi-soft class C Lot C44-1209-M4B 0.1, 1 mg/kg 8, 15hours

Results: FIG. 9 shows cell numbers in bronchoalveolar lavage fluid.Intranasal instillation of each of the class C ODNs, especially at 1mg/kg, showed a trend to causing a very mild accumulation of leukocytesin bronchoalveolar lavage fluid.

FIGS. 10-15 show cytokine concentrations in bronchoalveolar lavagefluid. Intranasal instillation of each of the CpG ODNs inducedmeasurable titers of IFNα, IFNγ, IP-10, IL-12p40, IL-6 and TNFα inbronchoalveolar lavage fluid. Titers of the other analytes measured didnot reach detectable concentrations or concentrations above background(typically <20 pg/ml, data not shown). The class C ODNs were each morepotent than the class B ODN SEQ ID NO:7 as inducers of IFNα, IFNγ,IP-10, IL-12p40, IL-6 and TNFα. The increased potency of the class CODNs was especially apparent at the 0.1 mg/kg dose level. Of particularinterest was the observation that only the class C ODNs were able toinduce any measurable titers of IFNα and IFNγ after dosing at 0.1 mg/kg(FIG. 10).

FIGS. 15-18 show cytokine concentrations in serum. Intranasalinstillation of each of the CpG ODNs induced measurable titers of IFNγ,IL-6 and TNFα in serum. Titers of the other analytes measured did notreach detectable concentrations (typically<20 pg/ml, data not shown).When compared with the class B CpG SEQ ID NO:7, each of the three classC ODNs were more potent inducers of the immune-modifying cytokines IFNα,IFNγ, IP-10, IL-12p40, IL-6 and TNFα.

Example 8 Effects of CpG Oligodeoxynucleotides SEQ ID NO:2 and SEQ IDNO:7 on Antigen-Induced IgE Production in the Mouse

Methods: Mice (male BALB/c) were sensitized on study days 0 and 7 withantigen (ovalbumin, 10 μg, i.p.) and aluminum hydroxide adjuvant PierceAlum, i.p.). Mice received SEQ ID NO:2 or SEQ ID NO:7 on study days −2,0, 5 and 7 (i.e. two days before each sensitization and on the day ofsensitization). Mice were bled by cardiac puncture on study day 18.Serum was collected by centrifugation and assayed by ELISA forovalbumin-specific IgE and IgG2a.

TABLE 8 Treatment groups: Sensitization Treatment n 1 None None 5 2Antigen Vehicle, i.p. 10 3 Antigen SEQ ID NO: 2, 1 μg/kg, i.p. 10 4Antigen SEQ ID NO: 2, 10 μg/kg, i.p. 10 5 Antigen SEQ ID NO: 2, 100μg/kg, i.p. 10 6 Antigen SEQ ID NO: 2, 1000 μg/kg, 10 i.p. 7 Antigen SEQID NO: 7, 1 μg/kg, i.p. 10 8 Antigen SEQ ID NO: 7, 10 μg/kg, i.p. 10 9Antigen SEQ ID NO: 7, 100 μg/kg, i.p. 10 10 Antigen SEQ ID NO: 7, 1000μg/kg, 10 i.p.

TABLE 9 Summary of study protocol Immunize Immunize ODN ODN ODN ODN Day:−2 0 5 7 18 Endpoints

Results: Antigen sensitization resulted in serum titers of antigen(ovalbumin)-specific IgE and IgG2a. The production of IgE was suppressedin mice treated with either SEQ ID NO:2 or SEQ ID NO:7, while theproduction of IgG2a was potentiated (FIG. 19).

Conclusions: The data of Example 8 demonstrates that SEQ ID NO:2 and SEQID NO:7 suppress Th2-associated production of IgE in response to antigensensitization, and potentiate Th1-associated IgG2a production. Theresults of this study provide further evidence that these CpG oligos cansuppress a Th2-type response to antigen exposure in the mouse.

Example 9 Effects of SEQ ID NO:2 Against Exacerbated AirwaysInflammation Induced by Combined Influenza Infection and AntigenChallenge

Introduction: The class C CpG oligodeoxynucleotide SEQ ID NO:2 cansuppress influenza virus load and virus-induced airways inflammation inmice. The present study investigated the protective effects of SEQ IDNO:2 against the exacerbated airways inflammation induced by combinedinfluenza virus infection and antigen challenge.

Methods: Antigen and virus administrations: Mice (male BALB/c) weresensitized on study days 0 and 7 with antigen (cockroach, 10 μg,intraperitoneal) with aluminum hydroxide adjuvant (Pierce Alum). Micewere antigen challenged by exposure to intranasally-administered antigen(10 μg in 40 μl saline), twice each week for three consecutive weeks.The first challenge was on study day 21. Mice were infected withinfluenza virus (influenza type A, subtype H1N1, mouse adapted strainPR8, 200 EID₅₀ in 40 μl saline) by intranasal instillation on study day34 (i.e. before the last pair of antigen challenges). Alternatively,separate groups of mice received antigen challenge alone or virusinfection alone.

SEQ ID NO:2 (100 μg/kg) was administered intranasally once each week,two days before the first antigen challenge of the week. Airwaysinflammation was assessed 48 hours after the last antigen challenge.Cells in airways were recovered by bronchoalveolar lavage. Differentialcell counts were made by light microscopy from cytocentrifugepreparations stained with Wright-Giemza stain.

TABLE 10 Summary of study protocol Antigen Antigen Antigen Antigensensitize ODN challenge ODN challenge ODN Virus challenge Day: 0 7 19 2124 26 28 31 33 34 35 38 40 First Second Third Endpoints treatment weektreatment week treatment week

Results: FIG. 20 shows that infection with influenza virus alone orantigen challenge alone each caused an increase in the total number ofleukocytes in bronchoalveolar lavage fluid. In virus-infected mice, thiscell accumulation included a marked neutrophilia, whereas inantigen-challenged mice, the accumulation included a markedeosinophilia. When compared with mice that received antigen challengealone, those that were antigen-challenged and virus-infected showed anexacerbated accumulation of leukocytes in bronchoalveolar lavage fluid.This increased accumulation included both neutrophils and mononuclearcells. However, these mice showed reduced eosinophilia. Otherresearchers have similarly shown that influenza infection can suppressairways eosinophilia in antigen-challenged mice, and have hypothesizedthat this is a Th1-mediated effect (e.g. Wohlleben et al., 2003).

Treatment with SEQ ID NO:2 (100 μg/kg) did not suppress thevirus-induced neutrophilia (FIG. 20). This negative finding was expectedsince, in an earlier study a higher dose of 300 μg/kg was most desirableto show anti-virus effects. Furthermore, SEQ ID NO:2 (100 μg/kg) didsignificantly suppress antigen-induced eosinophilia. This positivefinding was in agreement with earlier studies.

SEQ ID NO:2 (100 μg/kg) significantly suppressed the exacerbated airwaysinflammation induced in mice that were both virus-infected andantigen-challenged. The exacerbated accumulations of neutrophils andmononuclear cells were both suppressed. In addition to exacerbatedairways inflammation, mice that were both virus-infected andantigen-challenged showed a marked loss of body weight. This wassignificantly suppressed in mice treated with SEQ ID NO:2.

Conclusions: In both children and adults with existing asthma,infections with respiratory tract viruses are important precipitants forairway obstruction and wheezing. The inflammatory processes involved arecomplex. However, virus-induced neutrophil and mononuclear cellrecruitment and activation are implicated in aggravating the airwayobstruction that contributes to these asthma exacerbations (reviewed byGern and Busse, Nature Immunology, 2002). The data of Example 9demonstrate that SEQ ID NO:2 markedly suppresses the exacerbatedaccumulations of neutrophils and mononuclear cells induced in mice bycombined virus infection and antigen challenge.

Example 10 Guinea Pig Studies Example 10a Summary of Guinea Pig AHRProtocol

Male guinea pigs were sensitized on study days 0 and 4 with antigen(ovalbumin, 0.5 ml, 1% OVA i.p./s.c.) with aluminum hydroxide adjuvant.Guinea pigs were antigen challenged by exposure to inhaled ovalbuminaerosol, twice each week for two consecutive weeks. The first challengewas on study day 13. CpG ODN or vehicle (saline, 20 μl) wereadministered intranasally once each week, two days before the firstantigen challenge of the week. Airways hyperreactivity was assessed 24hours after the last antigen challenge by measuring bronchoconstriction(increase in airway resistance) to intravenous methacholine. For eachanimal, a dose-response curve to methacholine was obtained, and airwayreactivity was quantified as the area under the curve. FIG. 21 shows aschematic of the procedure.

Example 10b Effect of SEQ ID NO:7 on Airway Resistance and LungCompliance in Guinea Pigs

Method: Guinea pigs were sensitized as described in Example 10. Thefirst challenge was on study day 13. Guinea pigs were given intranasallyeither carrier (saline), OVA alone, of concentrations of SEQ ID NO:7 of10 μl/kg, 30 μl/kg, 100 μl/kg, or 300 μl/kg, i.t.

Results: FIG. 22 shows that SEQ ID NO:7 caused a dose-dependentreduction in AUC-resistance.

Example 10c Statistical Analysis of the Effect of SEQ ID NO:7 on AirwayResistance and Lung Compliance in Guinea Pigs

Method: The Dunnett multiple comparisons test was used to analyze thedata from the experiments in Example 10b. The Dunnett multiplecomparisons test allows comparison of all samples to a single controlgroup.

Results: FIG. 23 shows that SEQ ID NO:7 caused a dose-dependentreduction in AUC-resistance.

Example 10d Effect of SEQ ID NO:2 on Airway Resistance and LungCompliance in Guinea Pigs

Method: Guinea pigs were sensitized as described in Example 10. Thefirst challenge was on study day 13. Guinea pigs were given intranasallyeither carrier (saline), OVA alone, of concentrations of SEQ ID NO:2 of10 μl/kg, 30 μl/kg, 100 μl/kg, or 300 μl/kg, i.t.

Results: FIG. 24 shows that SEQ ID NO:2 caused a dose-dependentreduction in AUC-resistance.

Example 10e Statistical Analysis of the effect of SEQ ID NO:2 on AirwayResistance and Lung Compliance in Guinea Pigs

Method: The Dunnett multiple comparisons test was used to analyze thedata from the experiments in Example 10d. The Dunnett multiplecomparisons test allows comparison of all samples to a single controlgroup

Results: FIG. 25 shows that SEQ ID NO:2 caused a dose-dependentreduction in AUC-resistance.

Example 11

Levels of IL-10, TNF-alpha, interferon-gamma, and IL-6 (pg/ml) producedby human PBMC following exposure of these cells to the CpGoligonucleotides described herein is shown in the attached FIG. 27-31.The test oligonucleotides shown in FIG. 27 include SEQ ID NOs: 10, 9,13, 14, 1, and 2. The concentration of oligonucleotide used to produce aparticular data point is depicted along the X-axis (μM).

As demonstrated in FIG. 27 each of the oligonucleotides tested in theassays were able to produce different levels and patterns of IL-10secretion. Of those tested ODN SEQ ID NO 1 and 2 resulted indramatically higher induction of IL-10.

FIG. 28 depicts data relating to TNF-alpha, interferon-gamma, and IL-6at three representative doses. More detailed graphs on these cytokinesare depicted in FIGS. 29-31 with additional oligonucleotide dosages.

Example 12

Levels of B cell, plasmacytoid dendritic cell and monocyte activationfollowing exposure of these cells to the CpG oligonucleotides describedherein is shown in the attached FIGS. 32-42. The oligonucleotidesexamined are depicted in the Figures by SEQ ID NO and included SEQ IDNO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM).

As demonstrated in FIGS. 32, 34, 35, 40, and 42 the CpG oligonucleotidestested in the assays were able activate B cells, as represented by themarkers tested. In FIGS. 32 and 33 the CpG oligonucleotides tested inthe assays were able activate NK cells, as represented by the markerstested. In FIGS. 36, 37, 40, and 41 the CpG oligonucleotides tested inthe assays were able activate monocytes, as represented by the markerstested. In FIGS. 36 and 39 the CpG oligonucleotides tested in the assayswere able activate plasmacytoid dendritic cells, as represented by themarkers tested.

All five ODN having a semi soft backbone that were tested in the assaysshowed an increased potency in the assays (IFN-alpha, IP-10, IL-10)compared to SEQ ID NO. 9 (fully phosphorothioate backbone). The potencyof these semi-soft ODN is also increased in: Monocyte activation (CD80,CD86 expression), pDC activation (CD86 expression), Intracellular IP-10(Monocytes and B cells), IL-6 secretion, and B cell activation (CD80,CD86 expression). For instance, at approximately equivalent or lowerconcentrations most of the tested ODN resulted in better induction ofthe cell surface markers than the fully phosphorothioate SEQ ID NO. 9.

Example 13

The aim of this study was to investigate the biological activity ofselected fragments (putative metabolites) of SEQ ID NO: 2. Activity wasdetermined by measuring the ability of each fragment to induce secretionof TLR9-associated cytokines from mouse splenocytes in vivo.

Stimulation of cytokine secretion by fragments of SEQ ID NO.: 2 is shownin the attached FIGS. 43-45. The oligonucleotides examined are depictedin the Figures by SEQ ID NO and include SEQ ID NO: 15-23. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μg/ml).

As demonstrated in FIGS. 43-45 SEQ ID NO: 2 and the fragments (putativemetabolites, SEQ ID NOs:15-23) tested all induced the TLR9-associatedcytokines IFNα, IFNγ, IP-0, IL-6, IL-10 and TNFα from mouse splenocytesin vitro (FIGS. 43, 44 and 45). This data demonstrates that each of thefragments retained biological activity.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

1. A method for treating asthma, comprising administering to a subjecthaving or at risk of having asthma an oligonucleotide comprisingTCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO:2) in an effective amount to treatasthma.
 2. A method for treating asthma exacerbated by viral infection,comprising administering to a subject having or at risk of having asthmaexacerbated by viral infection an oligonucleotide comprisingTCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO:2), in an effective amount to treatthe asthma.
 3. The method of claim 2, wherein the oligonucleotide isadministered without an antigen to the subject.
 4. The method of claim1, wherein the oligonucleotide is delivered by a route selected from thegroup consisting of oral, nasal, sublingual, intravenous, subcutaneous,mucosal, respiratory, direct injection, and dermally.
 5. A method fortreating chronic obstructive pulmonary disease, comprising administeringto a subject having or at risk of having chronic obstructive pulmonarydisease an oligonucleotide comprising TCGTCGTCGTTCGGCGCGCGCCG (SEQ IDNO:2) in an effective amount to treat chronic obstructive pulmonarydisease.